Methods of using interleukin-3 (IL-3) mutant polypeptides for ex-vivo expansion of hematopoietic stem cells

Description

FIELD OF THE INVENTION

The present invention relates to mutants or variants of human interleukin-3 (hIL-3) which contain one or more amino acid substitutions and which may have portions of the native hIL-3 molecule deleted. These hIL-3 single and multiple mutation polypeptides retain one or more activities of native hIL-3 and may also show improved hematopoietic cell-stimulating activity and/or an improved activity profile which may include reduction of undesirable biological activities associated with native hIL-3.

BACKGROUND OF THE INVENTION

Colony stimulating factors (CSFs) which stimulate the differentiation and/or proliferation of bone marrow cells have generated much interest because of their therapeutic potential for restoring depressed levels of hematopoietic stem cell-derived cells. CSFs in both human and murine systems have been identified and distinguished according to their activities. For example, granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF) stimulate the in vitro formation of neutrophilic granulocyte and macrophage colonies, respectively while GM-CSF and interleukin-3 (IL-3) have broader activities and stimulate the formation of both macrophage, neutrophilic and eosinophilic granulocyte colonies. IL-3 also stimulates the formation of mast, megakaryocyte and pure and mixed erythroid colonies.

Because of its ability to stimulate the proliferation of a number of different cell types and to support the growth and proliferation of progenitor cells, IL-3 has potential for therapeutic use in restoring hematopoietic cells to normal amounts in those cases where the number of cells has been reduced due to diseases or to therapeutic treatments such as radiation and chemotherapy.

Interleukin-3 (IL-3) is a hematopoietic growth factor which has the property of being able to promote the survival, growth and differentiation of hematopoietic cells. Among the biological properties of IL-3 are the ability (a) to support the growth and differentiation of progenitor cells committed to all, or virtually all, blood cell lineages; (b) to interact with early multipotential stem cells; (c) to sustain the growth of pluripotent precursor cells; (d) to stimulate proliferation of chronic myelogenous leukemia (CML) cells; (e) to stimulate proliferation of mast cells, eosinophils and basophils; (f) to stimulate DNA synthesis by human acute myelogenous leukemia (AML) cells; (g) to prime cells for production of leukotrienes and histamines; (h) to induce leukocyte chemotaxis; and (i) to induce cell surface molecules needed for leukocyte adhesion.

Mature human interleukin-3 (hIL-3) consists of 133 amino acids. It has one disulfide bridge and two potential glycosylation sites (Yang, et al., CELL 47:3 (1986)).

Murine IL-3 (mIL-3) was first identified by Ihle, et al., J. IMMUNOL. 126:2184 (1981) as a factor which induced expression of a T cell associated enzyme, 20-hydroxysteroid dehydrogenase. The factor was purified to homogeneity and shown to regulate the growth and differentiation of numerous subclasses of early hematopoietic and lymphoid progenitor cells.

In 1984, cDNA clones coding for murine IL-3 were isolated (Fung, et al., NATURE 307:233 (1984) and Yokota, et al., PROC. NATL. ACAD. SCI. USA 81:1070 (1984)). The murine DNA sequence coded for a polypeptide of 166 amino acids including a putative signal peptide.

The gibbon IL-3 sequence was obtained using a gibbon cDNA expression library. The gibbon IL-3 sequence was then used as a probe against a human genomic library to obtain a human IL-3 sequence.

Gibbon and human genomic DNA homologues of the murine IL-3 sequence were disclosed by Yang, et al., CELL 47:3 (1986). The human sequence reported by Yang, et al. included a serine residue at position 8 of the mature protein sequence. Following this finding, others reported isolation of Pro

8

hIL-3 cDNAs having proline at position 8 of the protein sequence. Thus it appears that there may be two allelic forms of hIL-3.

Dorssers, et al., GENE 55:115 (1987), found a clone from a human cDNA library which hybridized with mIL-3. This hybridization was the result of the high degree of homology between the 3′ noncoding regions of mIL-3 and hIL-3. This cDNA coded for an hIL-3 (Pro

8

) sequence.

U.S. Pat. Nos. 4,877,729 and 4,959,455 disclose human IL-3 and gibbon IL-3 cDNAs and the protein sequences for which they code. The hIL-3 disclosed has serine rather than proline at position 8 in the protein sequence.

Clark-Lewis, et al., SCIENCE 231:134 (1986) performed a functional analysis of murine IL-3 analogues synthesized with an automated peptide synthesizer. The authors concluded that the stable tertiary structure of the complete molecule was required for full activity. A study on the role of the disulfide bridges showed that replacement of all four cysteines by alanine gave a molecule with {fraction (1/5000)}th the activity as the native molecule. Replacement of two of the four Cys residues by Ala(Cys

79

, Cys

140

→Ala

79

, Ala

140

) resulted in an increased activity. The authors concluded that in murine IL-3 a single disulfide bridge is required between cysteines 17 and 80 to get biological activity that approximates physiological levels and that this structure probably stabilizes the tertiary structure of the protein to give a conformation that is optimal for function. (Clark-Lewis, et al., PROC. NATL. ACAD. SCI. USA 85:7897 (1988)).

International Patent Application (PCT) WO 89/00598 discloses gibbon- and human-like IL-3. The hIL-3 contains a Ser

8

→Pro

8

replacement. Suggestions are made to replace Cys by Ser, thereby breaking the disulfide bridge, and to replace one or more amino acids at the glycosylation sites.

EP-A-0275598 (WO 88/04691) illustrates that Ala

1

can be deleted while retaining biological activity. Some mutant hIL-3 sequences are provided, e.g., two double mutants, Ala

1

→Asp

1

, Trp

13

→Arg

13

(pGB/IL-302) and Ala

1

→Asp

1

, Met

3

→Thr

3

(pGB/IL-304) and one triple mutant Ala

1

→Asp

1

, Leu

9

→Pro

9

, Trp

13

→Arg

13

(pGB/IL-303).

WO 88/05469 describes how deglycosylation mutants can be obtained and suggests mutants of Arg

54

Arg

55

and Arg

108

Arg

109

Lys

10

might avoid proteolysis upon expression in

Saccharomyces cerevisiae

by KEX2 protease. No mutated proteins are disclosed. Glycosylation and the KEX2 protease activity are only important, in this context, upon expression in yeast.

WO 88/06161 mentions various mutants which theoretically may be conformationally and antigenically neutral. The only actually performed mutations are Met

2

→Ile

2

and Ile

131

→Leu

131

. It is not disclosed whether the contemplated neutralities were obtained for these two mutations.

WO 91/00350 discloses nonglycosylated hIL-3 analog proteins, for example, hIL-3 (Pro

8

Asp

15

Asp

70

), Met

3

rhul-3 (Pro

8

Asp

15

Asp

70

); Thr

4

rhuL-3 (Pro

8

Asp

15

Asp

70

) and Thr

6

rhuIL-3 (Pro

8

Asp

15

Asp

70

). It is said that these protein compositions do not exhibit certain adverse side effects associated with native hIL-3 such as urticaria resulting from infiltration of mast cells and lymphocytes into the dermis. The disclosed analog hIL-3 proteins may have N termini at Met

3

, Thr

4

, or Thr

6

.

WO 90/12874 discloses cysteine added variants (CAVs) of IL-3 which have at least one Cys residue substituted for a naturally occurring amino acid residue.

SUMMARY OF THE INVENTION

The present invention relates to recombinant human interleukin-3 (hIL-3) variant or mutant proteins (muteins). These hIL-3 muteins contain amino acid substitutions and may also have amino acid deletions at either/or both the N- and C-termini. Preferably, these mutant polypeptides of the present invention contain one to three amino acids which differ from the amino acids found at the corresponding positions in the native hIL-3 polypeptide. The invention also relates to pharmaceutical compositions containing the hIL-3 muteins, DNA coding for the muteins, and methods for using the muteins. Additionally, the present invention relates to recombinant expression vectors comprising nucleotide sequences encoding the hIL-3 muteins, related microbial expression systems, and processes for making the hIL-3 muteins using the microbial expression systems.

The present invention includes mutants of hIL-3 in which from 1 to 14 amino acids have been deleted from the N-terminus and/or from 1 to 15 amino acids have been deleted from the C-terminus, and in which from one to three amino acid substitutions have been made. Preferred muteins of the present invention are those in which amino acids 1 to 14 have been deleted from the N-terminus, or amino acids 126 to 133 have been deleted from the C-terminus, and which both also contain from one to three amino acid substitutions in the polypeptide sequence. These hIL-3 multiple mutation polypeptides may have biological activities similar to or better than hIL-3 and, in some cases, may also have an improved side effect profile, i.e., some muteins may have a better therapeutic index than native hIL-3. The present invention also provides muteins which may function as IL-3 antagonists or as discrete antigenic fragments for the production of antibodies useful in immunoassay and immunotherapy protocols. In addition to the use of the hIL-3 mutant polypeptides of the present invention in vivo, it is envisioned that in vitro uses would include the ability to stimulate bone marrow and blood cell activation and growth before infusion into patients.

Antagonists of hIL-3 would be particularly useful in blocking the growth of certain cancer cells like AML, CML and certain types of B lymphoid cancers. Other conditions where antagonists would be useful include those in which certain blood cells are produced at abnormally high numbers or are being activated by endogenous ligands. Antagonists would effectively compete for ligands, presumably naturally occurring hemopoietins including and not limited to IL-3, GM-CSF and IL-5, which might trigger or augment the growth of cancer cells by virtue of their ability to bind to the IL-3 receptor complex while intrinsic activation properties of the ligand are diminished. IL-3, GM-CSF and or IL-5 also play a role in certain asthmatic responses. An antagonist of the IL-3 receptor may have utility in this disease by blocking receptor-mediated activation and recruitment of inflammatory cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

is the human IL-3 gene for

E. coli

expression (pMON5873), encoding the polypeptide sequence of natural (wild type) human IL-3 [SEQ ID NO:128], plus an initiator methionine, as expressed in

E. coli

, with the amino acids numbered from the N-terminus of the natural hIL-3.

FIG.

2

: ClaI to NsiI Replacement Fragment.

FIG. 2

shows the nucleotide sequence of the replacement fragment used between the ClaI and NsiI sites of the hIL-3 gene. The codon choice used in the fragment corresponds to that found in highly expressed

E. coli

genes (Gouy and Gautier, 1982). Three new unique restriction sites, EcoRV, XhoI and PstI were introduced for the purpose of inserting synthetic gene fragments. The portion of the coding sequence shown encodes hIL-3 amino acids 20-70.

FIGS. 3A and 3B

shows the nucleotide and amino acid sequence of the gene in pMON5873 with the sequence extending from NcoI through HindIII. The codon choices used to encode amino acids 1-14 and 107-133 correspond to that found in highly expressed

E. coli

genes.

FIG. 4

shows the construction of the plasmid vector pMON5846 which encodes [Met-(1-133) hIL-3 (Arg

129

)].

FIG. 5

shows the construction of the plasmid vector pMON5847 (ATCC 68912) which encodes [Met-(1-133) hIL-3 (Arg

129

)].

FIG. 6

shows the construction of plasmid vector pMON5853 which encodes [Met-(15-133) hIL-3 (Arg

129

)].

FIG. 7

shows the construction of the plasmid vector pMON5854 which encodes [Met-(1-133) hIL-3 (Arg

129

)].

FIG. 8

shows the DNA sequence and resulting amino acid sequence of the lamB signal peptide.

FIG. 9

shows the construction of the plasmid vector pMON5978 which encodes Met-Ala-(15-125) hIL-3.

FIG. 10

shows the construction of the plasmid vector pMON5988 which encodes Met-Ala(15-125) hIL-3.

FIG. 11

shows the construction of the plasmid vector pMON5887 which encodes Met-(1-125) hIL-3.

FIG. 12

shows the construction of pMON6457 which encodes (15-125) hIL-3; it contains the araBAD promoter and the lamB signal peptide fused to the variant hIL-3 amino acids 15-125.

FIG. 13

shows the construction of pMON6458; it contains the araBAD promoter and the lamb signal peptide fused to the variant hIL-3 amino acids 15-125.

FIG. 14

shows the construction of pMON6467 in which the bases encoding amino acids 35-40 of hIL-3 were deleted using site-directed PCR mutagenesis methods. pMON6467 was used as the template for the generation of single amino acid variants at positions 35-40 of hIL-3.

FIG. 15

shows the construction of single amino acid substitutions at position 35 of hIL-3 using site-directed PCR mutagenesis methods. The mutagenesis results in 20 different single amino substitutions, which is referred to as a “library”, at position 35 of hIL-3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to muteins of human interleukin-3 (hIL-3) in which amino acid substitutions have been made at from one to three positions in the amino acid sequence of the polypeptide and to hIL-3 muteins which have substantially the same structure and substantially the same biological activity. Preferred muteins of the present invention are (15-125) hIL-3 deletion mutants which have deletions of amino acids 1 to 14 at the N-terminus and/or 126 to 133 at the C-terminus and which both also have from one to three amino acid substitutions in the polypeptide and muteins having substantially the same structure and substantially the same biological activity. As used herein human interleukin-3 corresponds to the amino acid sequence (1-133) as depicted in FIG.

1

and (15-125) hIL-3 Corresponds to the 15 to 125 amino acid sequence of the hIL-3 polypeptide. Naturally occurring variants of hIL-3 polypeptide amino acids are also included in the present invention (for example, the allele in which proline rather than serine is at position 8 in the hIL-3 polypeptide sequence) as are variant hIL-3 molecules which are modified post-translationally (e.g. glycosylation).

The present invention also includes the DNA sequences which code for the mutant polypeptides, DNA sequences which are substantially similar and perform substantially the same function, and DNA sequences which differ from the DNAs encoding the muteins of the invention only due to the degeneracy of the genetic code.

Included in the present invention are novel mutant human interleukin-3 polypeptides comprising a polypeptide having the amino acid sequence of native human interleukin-3 wherein amino acids 126 to 133 have been deleted from the C-terminus of the native human interleukin-3 polypeptide and amino acids 1 to 14 have been deleted from the N-terminus of the native human interleukin-3 polypeptide and, in addition, polypeptides of the present invention also have one to three amino acid substitutions in the polypeptide sequence. The muteins of the present invention can have from one to three amino acid substitutions in the hIL-3 polypeptide chain and, in addition, can have deletions of amino acids at the N-terminus and/or the C-terminus.

Also included in the present invention are the DNA sequences coding for the muteins of the present invention; the oligonucleotide intermediates used to construct the mutant DNAs; and the polypeptides coded for by these oligonucleotides. These polypeptides may be useful as antagonists or as antigenic fragments for the production of antibodies useful in immunoassay and immunotherapy protocols.

The mutant hIL-3 polypeptides of the present invention may also have methionine, alanine, or methionine-alanine residues inserted at the N-terminus.

The present invention includes hIL-3 mutant polypeptides of the formula I:

Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn

[SEQ ID NO:15]

1 5 10 15

Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

20 25 30

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa

35 40 45

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

65 70 75

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

80 85 90

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

95 100 105

Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

110 115 120

Xaa Xaa Xaa Gln Gln Thr Thr Leu Ser Leu Ala Ile Phe

125 130

wherein

Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg:

Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln;

Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;

Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;

Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val;

Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly;

Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg;

Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu;

Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala;

Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp;

Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala;

Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp;

Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val;

Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys;

Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;

Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu;

Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu:

Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met;

Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val;

Xaa at position 36 is Asp, Leu, or Val;

Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile;

Xaa at position 38 is Asn, or Ala;

Xaa at position 40 is Leu, Trp, or Arg;

Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro;

Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala;

Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser;

Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro;

Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His;

Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly;

Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His;

Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn;

Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;

Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln;

Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;

Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr;

Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met;

Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His , Ala or Leu;

Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly;

Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys;

Xaa at position 57 is Asn or Gly;

Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;

Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg;

Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr;

Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;

Xaa at position 62 is Asn His, Val, Arg, Pro, Thr, Asp, or Ile;

Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;

Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys;

Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser;

Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;

Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His;

Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;

Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu;

Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala;

Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn;

Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;

Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;

Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala;

Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu:

Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp;

Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu;

Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;

Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;

Xaa at position 80 is Agn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;

Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys;

Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;

Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met;

Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val;

Xaa at position 85 is Leu, Asn, Val, or Gln;

Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys;

Xaa at position 87 is Leu, Ser, Trp, or Gly;

Xaa at position 88 is Ala, Lys, Arg, Val, or Trp;

Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser;

Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met;

Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;

Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu;

Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;

Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro;

Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr;

Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr;

Xaa at position 97 is Ile, Val, Lys, Ala, or Asn;

Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro;

Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His;

Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro;

Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu, or Gln;

Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro;

Xaa at position 103 is Asp, or Ser;

Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly;

Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro;

Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro;

Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly;

Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp;

Xaa at position 111 is Leu, Ile, Arg, Asp, or Met;

Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe;

Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn;

Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;

Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met;

Xaa at position 116 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile;

Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro;

Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;

Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;

Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln;

Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;

Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

and which can additionally have Met- preceding the amino acid in position 1; and wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3 with the proviso that when Xaa at position 22 is Leu, and/or Xaa at position 34 is Gly or Glu, and/or Xaa at position 44 is Ala, and/or Xda at position 46 is Lys or Ala, and/or Xaa at position 50 is Lys, and/or Xaa at position 59 is Pro or Arg, and/or Xaa at position 63 is Lys, and/or Xaa at position 75 is Gly or Arg, and/or Xaa at position 94 is Pro, and/or Xaa at position 98 is Arg, and/or Xaa at position 106 is Lys, and/or Xaa at position 110 is Ala or Glu, and/or Xaa at position 111 is Met, then there must be at least one additional substitution besides the ones indicated.

Included in the present invention are (1-133) hIL-3 mutant polypeptides of the Formula II:

Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn

(SEQ ID NO:16)

1 5 10 15

Cys Xaa Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa

20 25 30

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Glu Xaa Xaa

35 40 45

Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

65 70 75

Xaa Xaa Leu Xaa kaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa

80 85 90

Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa

95 100 105

Xaa Phe Xaa Xaa Lys Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Xaa Xaa

110 115 120

Xaa Xaa Xaa Gln Gln Thr Thr Leu Ser Leu Ala Ile Phe

125 130

wherein

Xaa at position 17 is Ser, Gly, Asp, Met, or Gln; Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln;

Xaa at position 19 is Met, Phe, Ile, Arg, or Ala;

Xaa at position 20 is Ile or Pro;

Xaa at position 21 is Asp or Glu;

Xaa at position 21 is Ile, Val, Ala, Leu, or Gly;

Xaa at position 24 is Ile, Val, Phe, or Leu;

Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala;

Xaa at position 26 is His, Phe, Gly, Arg, or Ala;

Xaa at position 28 is Lys, Leu, Gln, Gly, Pro, or Val;

Xaa at position 29 is Gln, Asn, Leu, Arg, or Val;

Xaa at position 30 is Pro, His, Thr, Gly, or Gln;

Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;

Xaa at position 32 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu;

Xaa at position 33 is Pro, Leu, Gln, Ala, or Glu;

Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;

Xaa at position 35 is Leu, Ala, Asn, Pro, Gln, or Val;

Xaa at position 36 is Asp or Leu;

Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile;

Xaa at position 38 is Asn or Ala;

Xaa at position 41 is Asn, Cys, Arg, His, Met, or Pro;

Xaa at position 42 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, Val or Arg;

Xaa at position 44 is Asp or Glu;

Xaa at position 45 is Gln, Val, Met, Leu, Thr, Lys, Ala, Asn, Glu, Ser, or Trp;

Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Ala, Asn, Gln, Glu, His, Ile, Lys, Tyr, Val or Gly;

Xaa at position 47 is Ile, Val, or His;

Xaa at position 49 is Met, Asn, or Asp;

Xaa at position 50 is Glu, Thr, Ala, Asn, Ser or Asp;

Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;

Xaa at position 52 is Asn or Gly;

Xaa at position 53 is Leu, Met, or Phe;

Xaa at position 54 is Arg, Ala, or Ser;

Xaa at position 55 is Arg, Thr, Val, Leu, or Gly;

Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Ala, Arg, Asn, Glu, His, Leu, Thr, Val or Lys;

Xaa at position 59 is Glu, Tyr, His, Leu, or Arg;

Xaa at position 60 is Ala, Ser, Asn, or Thr;

Xaa at position 61 is Phe or Ser;

Xaa at position 62 is Asn, Val, Pro, Thr, or Ile;

Xaa at position 63 is Arg, Tyr, Lys, Ser, His, or Val;

Xaa at position 64 is Ala or Asn;

Xaa at position 65 is Val, Thr, Leu, or Ser;

Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;

Xaa at position 67 is Ser, Phe, Val, Gly, Asn, Ile, or His:

Xaa at position 68 is Leu, Val, Ile, Phe, or His;

Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly;

Xaa at position 70 is Asn or Pro;

Xaa at position 71 is Ala, Met, Pro, Arg, Glu, Thr, or Gln;

Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;

Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, Arg, or Pro;

Xaa at position 74 is Ile or Met;

Xaa at position 75 is Glu, Gly, Asp, Ser, or Gln;

Xaa at position 76 is Ser, Val, Ala, Asn, Glu, Pro, Gly, or Asp;

Xaa at position 77 is Ile, Ser, or Leu;

Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;

Xaa at position 80 is Asn, Val, Gly, Thr, Leu, Glu, or Arg;

Xaa at position 81 is Leu, or Val;

Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;

Xaa at position 83 is Pro, Ala, Thr, Trp, or Met;

Xaa at position 85 is Leu or Val;

Xaa at position 87 is Leu or Ser;

Xaa at position 88 is Ala, Arg, or Trp;

Xaa at position 89 is Thr, Asp, Glu, His, Asn, or Ser;

Xaa at position 90 is Ala, Asp, or Met;

Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, or Asp;

Xaa at position 92 is Pro or Ser;

Xaa at position 93 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg;

Xaa at position 95 is His, Pro, Arg, Val, Leu, Gly, Asn, Ile, Phe, Ser or Thr;

Xaa at position 96 is Pro or Tyr;

Xaa at position 97 is Ile, Val, or Ala;

Xaa at position 98 is His, Ile, Asn, Asp, Ala, Thr, Leu, Arg, Gln, Glu, lys, Met, Ser, Tyr, Val or Pro;

Xaa at position 99 is Ile, Leu, Val, or Phe;

Xaa at position 100 is Lys, Leu, His, Arg, Ile, Gln, Pro, or Ser;

Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val, Asn, Ile, Leu or Tyr;

Xaa at position 102 is Gly, Glu, Lys, or Ser;

Xaa at position 104 is Trp, Val, Tyr, Met, or Leu;

Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

Xaa at position 106 is Glu, Ser, Ala, or Gly;

Xaa at position 108 is Arg, Ala, Gln, Ser or Lys;

Xaa at position 109 is Arg, Thr, Glu, Leu, Ser, or Gly;

Xaa at position 112 is Thr, Val, Gln, Glu, His, or Ser;

Xaa at position 114 is Tyr or Trp;

Xaa at position 115 is Leu or Ala;

Xaa at position 116 is Lys, Thr, Met, Val, Trp, Ser, Leu, Ala, Asn, Gln, His, Met, Phe, Tyr or Ile;

Xaa at position 117 is Thr, Ser, or Asn;

Xaa at position 119 is Glu, Ser, Pro, Leu, Thr, or Tyr;

Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln;

Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;

Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

and which can additionally have Met- preceding the amino acid in position 1; and wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3 with the proviso that when Xaa at position 34 is Gly or/and Xaa or position 46 is Lys or Ala or/and Xaa at position 59 is Arg and/or Xaa at position 63 is Lys and/or Xaa at position 75 is Gly and/or Xaa at position 98 is Arg then there must be at least one additional substitution besides the ones indicated.

Included in the present invention are (1-133) hIL-3 mutant polypeptides of the Formula III:

Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn

(SEQ ID NO:17)

1 5 10 15

Cys Xaa Xaa Xaa Ile Xaa Glu Xaa Xaa Xaa Xaa Leu Lys Xaa Xaa

20 25 30

Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Asn Leu Asn Xaa Glu Xaa Xaa

35 40 45

Xaa Ile Leu Met Xaa Xaa Asn Leu Xaa Xaa Xaa Asn Leu Glu Xaa

50 55 60

Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Ile Glu

65 70 75

Xaa Xaa Leu Xaa Xaa Leu Xaa Xaa Cys Xaa Pro Xaa Xaa Thr Ala

80 85 90

Xaa Pro Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Asp Xaa Xaa

95 100 105

Xaa Phe Xaa Xaa Lys Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Glu Xaa

110 115 120

Xaa Xaa Xaa Gln Gln Thr Thr Leu Ser Leu Ala Ile Phe

125 130

wherein

Xaa at position 17 is Ser, Gly, Asp, Met, or Gln;

Xaa at position 18 is Asn, His, or Ile;

Xaa at position 19 is Met or Ile;

Xaa at position 21 is Asp or Glu;

Xaa at position 23 is Ile, Ala, Leu, or Gly;

Xaa at position 24 is Ile, Val, or Leu;

Xaa at position 25 is Thr, His, Gln, or Ala;

Xaa at position 26 is His or Ala;

Xaa at position 29 is Gln, Asn, or Val;

Xaa at position 30 is Pro, Gly, or Gln;

Xaa at position 31 is Pro, Asp, Gly, or Gln;

Xaa at position 32 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu;

Xaa at position 33 is Pro or Glu;

Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;

Xaa at position 35 is Leu, Ala, Asn, Pro, Gln, or Val;

Xaa at position 37 is Phe, Ser, Pro, or Trp;

Xaa at position 38 is Asn or Ala;

Xaa at position 42 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr or Arg;

Xaa at position 44 is Asp or Glu;

Xaa at position 45 is Gln, Val, Met, Leu, Thr, Ala, Asn, Glu, Ser or Lys;

Xaa at position 46 is Asp, Phe, Ser, Thr, Ala, Asn Gln, Glu, His, Ile, Lys, Tyr, Val or Cys;

Xaa at position 50 is Glu, Ala, Asn, Ser or Asp;

Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His;

Xaa at position 54 is Arg or Ala;

Xaa at position 54 is Arg or Ala;

Xaa at position 55 is Arg, Thr, Val, Leu, or Gly;

Xaa at position 56 is Pro, Gly, Ser, Gln, Ala, Arg, Asn, Glu, Leu, Thr, Val or Lys;

Xaa at position 60 is Ala or Ser;

Xaa at position 62 is Asn, Pro, Thr, or Ile;

Xaa at position 63 is Arg or Lys;

Xaa at position 64 is Ala or Asn;

Xaa at position 65 is Val or Thr;

Xaa at position 66 is Lys or Arg;

Xaa at position 67 is Ser, Phe, or His;

Xaa at position 68 is Leu, Ile, Phe, or His;

Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly;

Xaa at position 71 is Ala, Pro, or Arg;

Xaa at position 72 is Ser, Glu, Arg, oz Asp;

Xaa at position 73 is Ala or Leu;

Xaa at position 76 is Ser, Val, Ala, Asn, Glu, Pro, or Gly;

Xaa at position 77 is Ile or Leu;

Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;

Xaa at position 80 is Asn, Gly, Glu, or Arg;

Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Ile, Met, Phe, Ser, Thr, Tyr or Val;

Xaa at position 83 is Pro or Thr;

Xaa at position 85 is Leu or Val;

Xaa at position 87 is Leu or Ser;

Xaa at position 88 is Ala or Trp;

Xaa at position 91 is Ala or Pro;

Xaa at position 93 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg;

Xaa at position 95 is His, Pro, Arg, Val, Leu, Gly, Asn, Phe, Ser or Thr;

Xaa at position 96 is Pro or Tyr;

Xaa at position 97 is Ile or Val;

Xaa at position 98 is His, Ile, Asn, Leu, Ala, Thr, Arg, Gln, Lys, Met, Ser, Tyr, Val or Pro;

Xaa at position 99 is Ile, Leu, or Val;

Xaa at position 100 is Lys, Arg, Ile, Gln, Pro, or Ser;

Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Pro, Asn, Ile, Leu or Tyr;

Xaa at position 104 is Trp or Leu;

Xaa at position 105 is Asn, Pro, Ala, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

Xaa at position 106 is Glu or Gly;

Xaa at position 108 is Arg, Ala, or Ser;

Xaa at position 109 is Arg, Thr, Glu, Leu, or Ser;

Xaa at position 112 is Thr, Val, or Gln;

Xaa at position 114 is Tyr or Trp;

Xaa at position 115 is Leu or Ala;

Xaa at position 116 is Lys, Thr, Val, Trp, Ser, Ala, His, Met, Phe, Tyr or Ile;

Xaa at position 117 is Thr or Ser;

Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln;

Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Asp, or Gly;

Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

and which can additionally have Met- preceding the amino acid in position 1; and wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3 with the proviso that when Xaa at position 22 is Leu, and/or Xaa at position 34 is Gly or Glu, and/or Xaa at position 44 is Ala, and/or Xaa at position 46 is Lys or Ala, and/or Xaa at position 50 is Lys, and/or Xaa at position 59 is Pro or Arg, and/or Xaa at position 63 is Lys, and/or Xaa at position 75 is Gly or Arg, and/or Xaa at position 94 is Pro, and/or Xaa at position 98 is Arg, and/or Xaa at position 106 is Lys, and/or Xaa at position 110 is Ala or Glu, and/or Xaa at position 111 is Met, then there must be at least one additional substitution besides the ones indicated. and which can additionally have Met- preceding the amino acid in position 1; and wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3 with the proviso that when Xaa at position 34 is Gly and/or Xaa at position 46 is Lys or Ala, and/or Xaa at position 63 is Lys, and/or Xaa at position 98 is Arg, then two or three of the amino acid designated by Xaa are different from the corresponding amino acids of the native (1-133) human interleukin-3.

Included in the present invention are (1-133) hIL-3 mutant polypeptides of the Formula IV:

Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn

[SEQ ID NO:18]

1 5 10 15

Cys Xaa Xaa Met Ile Asp Glu Xaa Ile Xaa Xaa Leu Lys Xaa Xaa

20 25 30

Pro Xaa Pro Xaa Xaa Asp Phe Xaa Asn Leu Asn Xaa Glu Asp Xaa

35 40 45

Xaa Ile Leu Met Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Glu Ala

50 55 60

Phe Xaa Arg Xaa Xaa Lys Xaa Xaa Xaa Asn Ala Ser Ala Ile Glu

65 70 75

Xaa Xaa Leu Xaa Xaa Leu Xaa Pro Cys Leu Pro Xaa Xaa Thr Ala

80 85 90

Xaa Pro Xaa Arg Xaa Pro Ile Xaa Xaa Xaa Xaa Gly Asp Trp Xaa

95 100 105

Glu Phe Xaa Xaa Lys Leu Xaa Phe Tyr Leu Xaa Xaa Leu Glu Xaa

110 115 120

Xaa Xaa Xaa Gln Gln Thr Thr Leu Ser Leu Ala Ile Phe

125 130

wherein

Xaa at position 17 is Ser, Gly, Asp, or Gln;

Xaa at position 18 is Asn, His, or Ile;

Xaa at position 23 is Ile, Ala, Leu, or Gly;

Xaa at position 25 is Thr, His, or Gln;

Xaa at position 26 is His or Ala;

Xaa at position 29 is Gln or Asn;

Xaa at position 30 is Pro or Gly;

Xaa at position 32 is Leu, Arg, Asn, or Ala;

Xaa at position 34 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr, or Met;

Xaa at position 35 is Leu, Ala, Asn, or Pro;

Xaa at position 29 is Asn or Ala;

Xaa at position 42 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr or Arg;

Xaa at position 45 is Gln, Val, Met, Leu, Ala, Asn, Glu, or Lys;

Xaa at position 46 is Asp, Phe, Ser, Ala, Gln, Glu, His, Val or Thr;

Xaa at position 50 is Glu Asn, Ser or Asp;

Xaa at position 51 is Asn, Arg, Pro, Thr, or His;

Xaa at position 55 is Arg, Leu, or Gly;

Xaa at position 56 is Pro, Gly, Ser, Ala, Asn, Val, Leu or Gln;

Xaa at position 62 is Asn, Pro, or Thr;

Xaa at position 64 is Ala or Asn;

Xaa at position 65 is Val or Thr;

Xaa at position 67 is Ser or Phe;

Xaa at position 68 is Leu or Phe;

Xaa at position 69 is Gln, Ala, Glu, or Arg;

Xaa at position 76 is Ser, Val, Asn, Pro, or Gly;

Xaa at position 77 is Ile or Leu;

Xaa at position 79 is Lys, Asn, Met, Arg, Ile, or Gly;

Xaa at position 80 is Asn, Gly, Glu, or Arg;

Xaa at position 82 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;

Xaa at position 87 is Leu or Ser;

Xaa at position 88 is Ala or Trp;

Xaa at position 91 is Ala or Pro;

Xaa at position 93 is Thr, Asp, or Ala;

Xaa at position 95 is His, Pro, Arg, Val, Gly, Asn, Ser or Thr;

Xaa at position 98 is His, Ile, Asn, Ala, Thr, Arg, Gln, Glu, Lys, Met, Ser, Tyr, Val or Leu;

Xaa at position 99 is Ile or Leu;

Xaa at position 100 is Lys or Arg;

Xaa at position 101 is Asp, Pro, Met, Lys, Thr, His, Pro, Asn, Ile, Leu or Tyr;

Xaa at position 105 is Asn, Pro, Ser, Ile or Asp;

Xaa at position 108 is Arg, Ala, or Ser;

Xaa at position 109 is Arg, Thr, Glu, Leu, or Ser;

Xaa at position 112 is Thr or Gln;

Xaa at position 116 is Lys, Val, Trp, Ala, His, Phe, Tyr or Ile;

Xaa at position 117 is Thr or Ser;

Xaa at position 120 is Asn, Pro, Leu, His, Val, or Gln;

Xaa at position 121 is Ala, Ser, Ile, Pro, or Asp;

Xaa at position 122 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr;

Xaa at position 123 is Ala, Met, Glu, Ser, or Leu;

and which can additionally have Met- preceding the amino acid in position 1; and wherein from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3.

Preferred polypeptides of the present invention are (15-125) hIL-3 mutant polypeptides of the Formula V:

Asn Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

[SEQ ID NO:19]

1 5 10 15

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa

20 25 30

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

35 40 45

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

65 70 75

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

80 85 90

Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

95 100 105

Xaa Xaa Xaa Xaa Gln Gln

110

wherein

wherein

Xaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg;

Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln;

Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys;

Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala;

Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val;

Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly;

Xaa at position 9 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg;

Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu;

Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala;

Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp;

Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or Ala;

Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp;

Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val;

Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys;

Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;

Xaa at position 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu;

Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu;

Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met;

Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val;

Xaa at position 22 is Asp, Leu, or Val;

Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile;

Xaa at position 24 is Asn, or Ala;

Xaa at position 26 is Leu, Trp, or Arg;

Xaa at position 27 is Asn, Cys, Arg, Leu, His, Met, Pro;

Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, Phe, Tyr, Ile or Met;

Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser;

Xaa at position 30 is Asp, Ser, Leu, Arg, Lys, Thr,Met, Trp, Glu, Asn, Gln, Ala or Pro;

Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, Ser, Ala, Ile, Glu, His or Trp;

Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly;

Xaa at position 33 is Ile, Gly, Val, Ser, Arg, Pro, or His;

Xaa at position 34 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn;

Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp;

Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln;

Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His;

Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr;

Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, Met, or;

Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu;

Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly;

Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys;

Xaa at position 43 is Asn or Gly;

Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys;

Xaa at position 45 is Glu Tyr, His, Leu, Pro, or Arg;

Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr;

Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser;

Xaa at position 48 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile;

Xaa at position 49 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val;

Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys;

Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser;

Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;

Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His;

Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His;

Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu;

Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala;

Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn;

Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;

Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg;

Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala;

Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu;

Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp;

Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu;

Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg;

Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp;

Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg;

Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys;

Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val;

Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met;

Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val;

Xaa at position 71 is Leu, Asn, Val, or Gln;

Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys;

Xaa at position 73 is Leu, Ser, Trp, or Gly;

Xaa at position 74 is Ala, Lys, Arg, Val, or Trp;

Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser;

Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met;

Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His;

Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu;

Xaa at position 79 iS Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg;

Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala or Pro;

Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile or Tyr;

Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr:

Xaa at position 83 is Ile, Val, Lys, Ala, or Asn;

Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro;

Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His;

Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, Pro;

Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu or Gln;

Xaa at position 88 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro;

Xaa at position 89 is Asp, or Ser;

Xaa at position 90 Is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly;

Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro;

Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro;

Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly;

Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala or Trp;

Xaa at position 97 is Leu, Ile, Arg, Asp, or Met;

Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe;

Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lsy, Leu, Ile, Val or Asn;

Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu;

Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met;

Xaa at position 102 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile;

Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro;

Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr;

Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg;

Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val, or Gln;

Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;

Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

and which can additionally have Met- or Met-Ala- preceding the amino acid in position 1; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding native amino acids of (1-133) human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

Included in the present invention are (15-125) hIL-3 mutant polypeptides of the Formula VI:

Asn Cys Xaa Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu Xaa Xaa

[SEQ ID NO:20]

1 5 10 15

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Glu Xaa

20 25 30

Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa

35 40 45

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55 60

Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa

65 70 75

Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa

80 85 90

Xaa Xaa Phe Xaa Xaa Lys Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Xaa

95 100 105

Xaa Xaa Xaa Xaa Gln Gln

110

wherein

Xaa at position 3 is Ser, Gly, Asp, Met, or Gln;

Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln;

Xaa at position 5 is Met, Phe, Ile, Arg, or Ala;

Xaa at position 6 is Ile or Pro;

Xaa at position 7 is Asp, or Glu;

Xaa at position 9 is Ile, Val, Ala, Leu, or Gly;

Xaa at position 10 is Ile, Val, Phe, or Leu;

Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala;

Xaa at position 12 is His, Phe, Gly, Arg, or Ala;

Xaa at position 14 is Lys, Leu, Gln, Gly, Pro, or Val;

Xaa at position 15 is Gln, Asn, Leu, Arg, or Val;

Xaa at position 16 is Pro, His, Thr, Gly, or Gln;

Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln;

Xaa at position 18 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu;

Xaa at position 19 is Pro, Leu, Gln, Ala, or Glu;

Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;

Xaa at position 21 is Leu, Ala, Asn, Pro, Gln, or Val;

Xaa at position 22 is Asp or Leu;

Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile;

Xaa at position 24 is Asn or Ala;

Xaa at position 27 is Asn, Cys, Arg, His, Met, or Pro;

Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, or Arg;

Xaa at position 30 is Asp, or Glu:

Xaa at position 31 is Gln, Val, Met, Leu, Thr, Lys, Ala, Asn Glu, Ser or Trp;

Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Ala, Asn, Gln, Glu, His, Ile, Lys, Tyr, Val or Gly;

Xaa at position 33 is Ile, Val, or His;

Xaa at position 35 is Met, Asn, or Asp;

Xaa at position 36 is Glu, Thr, Ala, Asn, Ser or Asp;

Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His;

Xaa at position 38 is Asn or Gly;

Xaa at position 39 is Leu, Met, or Phe;

Xaa at position 40 is Arg, Ala or Ser;

Xaa at position 41 is Arg, Thr, Val, Leu, or Gly;

Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Ala, Arg, Asn, Glu, His, Leu, Thr, Val or Lys;

Xaa at position 45 is Glu, Tyr, His, Leu, or Arg;

Xaa at position 46 is Ala, Ser, Asn, or Thr;

Xaa at position 47 is Phe or Ser;

Xaa at position 48 is Asn, Val, Pro, Thr, or Ile;

Xaa at position 49 is Arg, Tyr, Lys, Ser, His, or Val;

Xaa at position 50 is Ala or Asn;

Xaa at position 51 is Val, Thr, Leu, or Ser;

Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser;

Xaa at position 53 is Ser, Phe, Val, Gly, Asn, Ile, or His;

Xaa at position 54 is Leu, Val, Ile, Phe, or His;

Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly;

Xaa at position 56 is Asn or Pro;

Xaa at position 57 is Ala, Met, Pro, Arg, Glu, Thr, or Gln;

Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp;

Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, Arg, or Pro;

Xaa at position 60 is Ile or Met;

Xaa at position 61 is Glu, Gly, Asp, Ser, or Gln;

Xaa at position 62 is Ser, Val, Ala, Asn, Glu, Pro, Gly, or Asp;

Xaa at position 63 is Ile, Ser, or Leu;

Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp;

Xaa at position 66 is Asn, Val, Gly, Thr, Leu, Glu, or Arg;

Xaa at position 67 is Leu, or Val;

Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;

Xaa at position 69 is Pro, Ala, Thr, Trp, or Met;

Xaa at position 71 is Leu or Val;

Xaa at position 73 is Leu or Ser;

Xaa at position 74 is Ala, Arg, or Trp:

Xaa at position 75 is Thr, Asp, Glu, His, Asn, or Ser;

Xaa at position 76 is Ala, Asp, or Met;

Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, or Asp;

Xaa at position 78 is Pro or Ser;

Xaa at position 79 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg;

Xaa at position 81 is His, Pro, Arg, Val, Leu, Gly, Asn, Ile, Phe, Ser or Thr;

Xaa at position 82 is Pro or Tyr;

Xaa at position 83 is Ile, Val, or Ala;

Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Arg, Gln, Glu, Lys, Met, per, Tyr, Val or Pro;

Xaa at position 85 is Ile, Leu, Val, or Phe;

Xaa at position 86 is Lys, Leu, His, Arg, Ile, Gln, Pro or Ser;

Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Val, Asn, Ile, Leu or Tyr;

Xaa at position 99 is Gly, Glu, Lys, or Ser;

Xaa at position 90 is Trp, Val, Tyr, Met, or Leu;

Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

Xaa at position 92 is Glu, Ser, Ala, or Gly;

Xaa at position 94 is Arg, Ala, Gln, Ser or Lys;

Xaa at position 95 is Arg, Thr, Glu, Leu, Ser, or Gly;

Xaa at position 98 is Thr, Val, Gln, Glu, His, or Ser;

Xaa at position 100 is Tyr or Trp;

Xaa at position 101 is Leu or Ala;

Xaa at position 102 is Lys, Thr, Met, Val, Trp, Ser, Leu, Ala, Asn, Gln, His, Met, Phe, Tyr or Ile;

Xaa at position 103 is Thr, Ser, or Asn;

Xaa at position 105 is Glu, Ser, Pro, Leu, Thr, or Tyr;

Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln;

Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly;

Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

and which can additionally have Met- or Met-Ala- preceding the amino acid in position 1; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

Included in the present invention are (15-125) hIL-3 mutant polypeptides of the Formula VII:

Asn Cys Xaa Xaa Xaa Ile Xaa Glu Xaa Xaa Xaa Xaa Leu Lys Xaa

[SEQ ID NO:21]

1 5 10 15

Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Asn Leu Asn Xaa Glu Xaa

20 25 30

Xaa Xaa Ile Leu Met Xaa Xaa Asn Leu Xaa Xaa Xaa Asn Leu Glu

35 40 45

Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Ile

50 55 60

Glu Xaa Xaa Leu Xaa Xaa Leu Xaa Xaa Cys Xaa Pro Xaa Xaa Thr

65 70 75

Ala Xaa Pro Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Asp Xaa

80 85 90

Xaa Xaa Phe Xaa Xaa Lys Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Glu

95 100 105

Xaa Xaa Xaa Xaa Gln Gln

110

wherein

Xaa at position 3 is Ser, Gly, Asp, Met, or Gln;

Xaa at position 4 is Asn, His, or Ile;

Xaa at position 5 is Met or Ile;

Xaa at position 7 is Asp or Glu;

Xaa at position 9 is Ile, Ala, Leu, or Gly;

Xaa at position 10 is Ile, Val, or Leu;

Xaa at position 11 is Thr, His, Gln, or Ala;

Xaa at position 12 is His or Ala;

Xaa at position 15 is Gln, Asn, or Val;

Xaa at position 16 is Pro, Gly, or Gln;

Xaa at position 17 is Pro, Asp, Gly, or Gln;

Xaa at position 18 is Leu, Arg, Gln, Asn, Gly, Ala, or Glu;

Xaa at position 19 is Pro or Glu;

Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;

Xaa at position 21 is Leu, Ala, Asn, Pro, Gln, or Val;

Xaa at position 23 is Phe, Ser, Pro, or Trp;

Xaa at position 24 is Asn or Ala;

Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met Tyr or Arg;

Xaa at position 30 is Asp or Glu;

Xaa at position 31 is Gln, Val, Met, Leu, Thr, Ala, Asn, Glu, Ser or Lys;

Xaa at position 32 is Asp, Phe, Ser, Thr, Ala, Asn, Gln, Glu, His, Ile, Lys, Tyr, Val or Cys;

Xaa at position 36 is Glu, Ala, Asn, Ser or Asp;

Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His;

Xaa at position 40 is Arg or Ala;

Xaa at position 41 is Arg, Thr, Val, Leu, or Gly;

Xaa at position 42 is Pro, Gly, Ser, Gln, Ala, Arg, Asn, Glu, Leu, Thr, Val or Lys;

Xaa at position 46 is Ala or Ser;

Xaa at position 48 is Asn, Pro, Thr, or ale;

Xaa at position 49 is Arg or Lys;

Xaa at position 50 is Ala or Asn;

Xaa at position 51 is Val or Thr;

Xaa at position 52 is Lys or Arg;

Xaa at position 53 is Ser, Phe, or His;

Xaa at position 54 is Leu, Ile, Phe, or His;

Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, or Gly;

Xaa at position 57 is Ala, Pro, or Arg;

Xaa at position 58 is Ser, Glu, Arg, or Asp;

Xaa at position 59 is Ala or Leu;

Xaa at position 62 is Ser, Val, Ala, Asn, Glu, Pro, or Gly;

Xaa at position 63 is Ile or Leu;

Xaa at position 65 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp;

Xaa at position 66 is Asn, Gly, Glu, or Arg;

Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Ile, Met, Phe, Ser, Thr, Tyr or Val;

Xaa at position 69 is Pro or Thr;

Xaa at position 71 is Leu or Val;

Xaa at position 73 is Leu or Ser;

Xaa at position 74 is Ala or Trp;

Xaa at position 77 is Ala or Pro;

Xaa at position 79 is Thr, Asp, Ser, Pro, Ala, Leu, or Arg;

Xaa at position 81 is His, Pro, Arg, Val, Leu, Gly, Asn, Phe, Ser or Thr;

Xaa at position 82 is Pro or Tyr;

Xaa at position 83 is Ile or Val;

Xaa at position 84 is His, Ile, Asn, Ala, Thr, Leu, Arg, Gln, Lys, Met, Ser, Tyr, Val or Pro;

Xaa at position 85 is Ile, Leu, or Val;

Xaa at position 86 is Lys, Arg, Ile, Gln, Pro, or Ser;

Xaa at position 87 is Asp, Pro, Met, Lys, His, Thr, Asn, Ile, Leu or Tyr;

Xaa at position 90 is Trp or Leu;

Xaa at position 91 is Asn, Pro, Ala, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His;

Xaa at position 92 is Glu, or Gly;

Xaa at position 94 is Arg, Ala, or Ser;

Xaa at position 95 iS Arg, Thr, Glu, Leu, or Ser;

Xaa at position 98 is Thr, Val, or Gln;

Xaa at position 100 is Tyr or Trp;

Xaa at position 101 is Leu or Ala;

Xaa at position 102 is Lys, Thr, Val, Trp, Ser, Ala, His, Met, Phe, Tyr or Ile;

Xaa at position 103 is Thr or Ser;

Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln;

Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Asp, or Gly;

Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys;

Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu;

which can additionally have Met- or Met-Ala- preceding the amino acid in position 1; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (15-125) human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

Included in the present invention are (15-125) hIL-3 mutant polypeptides of the Formula VIII:

Asn Cys Xaa Xaa Met Ile Asp Glu Xaa Ile Xaa Xaa Leu Lys Xaa

[SEQ ID NO:22]

1 5 10 15

Xaa Pro Xaa Pro Xaa Xaa Asp Phe Xaa Asn Leu Asn Xaa Glu Asp

20 25 30

Xaa Xaa Ile Leu Met Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Glu

35 40 45

Ala Phe Xaa Arg Xaa Xaa Lys Xaa Xaa Xaa Asn Ala Ser Ala Ile

50 55 60

Glu Xaa Xaa Leu Xaa Xaa Leu Xaa Pro Cys Leu Pro Xaa Xaa Thr

65 70 75

Ala Xaa Pro Xaa Arg Xaa Pro Ile Xaa Xaa Xaa Xaa Gly Asp Trp

80 85 90

Xaa Glu Phe Xaa Xaa Lys Leu Xaa Phe Tyr Leu Xaa Xaa Leu Glu

95 100 105

Xaa Xaa Xaa Xaa Gln Gln

110

wherein

Xaa at position 3 is Ser, Gly, Asp, or Gln;

Xaa at position 4 is Asn, His, or Ile;

Xaa at position 9 is Ile, Ala, Leu, or Gly;

Xaa at position 11 is Thr, His, or Gln;

Xaa at position 12 is His or Ala;

Xaa at position 15 is Gln or Asn;

Xaa at position 16 is Pro or Gly;

Xaa at position 18 is Leu, Arg, Asn, or Ala;

Xaa at position 20 is Leu, Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr or Met;

Xaa at position 21 is Leu, Ala, Asn, or Pro;

Xaa at position 24 is Asn or Ala:

Xaa at position 28 is Gly, Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr or Arg;

Xaa at position 31 is Gln, Val, Met, Leu, Ala, Asn, Glu or Lys;

Xaa at position 32 is Asp, Phe, Ser, Ala, Gln, Glu, His, Val or Thr;

Xaa at position 36 is Glu, Asn, Ser or Asp;

Xaa at position 37 is Asn, Arg, Pro, Thr, or His;

Xaa at position 41 is Arg, Leu, or Gly;

Xaa at position 42 is Pro, Gly, Ser, Ala, Asn, Val, Leu or Gln;

Xaa at position 48 is Asn, Pro, or Thr;

Xaa at position 50 is Ala or Asn;

Xaa at position 51 is Val or Thr;

Xaa at position 53 is Ser or Phe;

Xaa at position 54 is Leu or Phe;

Xaa at position 55 is Gln, Ala, Glu, or Arg;

Xaa at position 62 is Ser, Val, Asn, Pro, or Gly;

Xaa at position 63 is Ile or Leu;

Xaa at position 65 is Lys, Asn, Met, Arg, Ile, or Gly;

Xaa at position 66 is Asn, Gly, Glu, or Arg;

Xaa at position 68 is Leu, Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser, Thr, Tyr or Val;

Xaa at position 73 is Leu or Ser;

Xaa at position 74 is Ala or Trp;

Xaa at position 77 is Ala or Pro;

Xaa at position 79 is Thr, Asp, or Ala;

Xaa at position 81 is His, Pro, Arg, Val, Gly, Asn, Ser or Thr;

Xaa at position 84 is His, Ile, Asn, Ala, Thr, Arg, Gln, Glu, Lys, Met, Ser, Tyr, Val or Leu;

Xaa at position 85 is Ile or Leu;

Xaa at position 86 is Lys or Arg;

Xaa at position 87 is Asp, Pro, Met, Lys, His, Pro, Asn, Ile, Leu or Tyr;

Xaa at position 91 is Asn, Pro, Ser, Ile or Asp;

Xaa at position 94 is Arg, Ala, or Ser;

Xaa at position 95 is Arg, Thr, Glu, Leu, or Ser;

Xaa at position 98 is Thr or Gln;

Xaa at position 102 is Lys, Val, Trp, or Ile;

Xaa at position 103 is Thr, Ala, His, Phe, Tyr or Ser;

Xaa at position 106 is Asn, Pro, Leu, His, Val, or Gln;

Xaa at position 107 is Ala, Ser, Ile, Pro, or Asp;

Xaa at position 108 is Gln, Met, Trp, Phe, Pro, His, Ile, or Tyr;

Xaa at position 109 is Ala, Met, Glu, Ser, or Leu;

and which can additionally have Met- or Met-Ala- preceding the amino acid in position 1; and wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133)human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

In Formulas V, VI, VII and VIII the Asn in position 1 corresponds to the Asn in position 15 of native hIL-3 and positions 1 to 111 correspond to positions 15 to 125 in the native hIL-3 sequence shown in FIG.

1

.

Also included in the present invention are polypeptides of the following formula (IX):

1 5 10

[SEQ ID NO:129]

(Met)

m

-Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr

15 20

Ser Trp Val Asn Cys Ser Xaa Met Ile Asp Glu Ile Ile

25 30 35

Xaa His Leu Lys Xaa Pro Pro Xaa Pro Leu Leu Asp Xaa

40 45 50

Asn Asn Leu Asn Xaa Glu Asp Xaa Asp Ile Leu Met Glu

55 60

Xaa Asn Leu Arg Xaa Pro Asn Leu Xaa Xaa Phe Xaa Arg

65 70 75

Ala Val Lys Xaa Leu Xaa Asn Ala Ser Xaa Ile Glu Xaa

80 85

Ile Leu Xaa Asn Leu Xaa Pro Cys Leu Pro Xaa Ala Thr

90 95 100

Ala Ala Pro Xaa Arg His Pro Ile Xaa Ile Lys Xaa Gly

105 110 115

Asp Trp Xaa Glu Phe Arg Xaa Lys Leu Thr Phe Tyr Leu

120 125

Xaa Thr Leu Glu Xaa Ala Gln Xaa Gln Gln Thr Thr Leu

130

Ser Leu Ala Ile Phe

wherein m is 0 or 1; Xaa at position 18 is Asn or Ile; Xaa at position 25 is Thr or His; Xaa at position 29 is Gln, Arg, or Val; Xaa at position 32 is Leu, Ala, or Asn; Xaa at position 37 is Phe, Pro, or Ser; Xaa at position 42 is Glu, Ala, or Ser; Xaa at position 45 is Gln, Val, or Met; Xaa at position 51 is Asn or Arg; Xaa at position 55 is Arg, Leu, or Thr; Xaa at position 59 is Glu or Leu; Xaa at position 60 is Ala or Ser; Xaa at position 62 is Asn or Val; Xaa at position 67 is Ser, Asn, or His; Xaa at position 69 is Gln or Glu; Xaa at position 73 is Ala or Gly; Xaa at position 76 is Ser or Ala; Xaa at position 79 is Lys or Arg; Xaa at position 82 is Leu, Glu, or Val; Xaa at position 87 is Leu or Ser; Xaa at position 93 is Pro or Ser; Xaa at position 98 is His, Ile, or Thr; Xaa at position 101 is Asp or Ala; Xaa at position 105 is Asn or Glu; Xaa at position 109 is Arg or Glu; Xaa at position 116 is Lys or Val; Xaa at position 120 is Asn, Gln, or His; Xaa at position 123 is Ala or Glu; wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

Polypeptides of the present invention include those (15-125) hIL-3 muteins of the following formula (X):

1 5 10

[SEQ ID NO:130]

(Met

m

-Ala

n

)p-Asn Cys Ser Xaa Met Ile Asp Glu Ile Ile

15 20

Xaa His Leu Lys Xaa Pro Pro Xaa Pro Leu Leu Asp Xaa

25 30 35

Asn Asn Leu Asn Xaa Glu Asp Xaa Asp Ile Leu Met Glu

40 45

Xaa Asn Leu Arg Xaa Pro Asn Leu Xaa Xaa Phe Xaa Arg

50 55 60

Ala Val Lys Xaa Leu Xaa Asn Ala Ser Xaa Ile Glu Xaa

65 70 75

Ile Leu Xaa Asn Leu Xaa Pro Cys Leu Pro Xaa Ala Thr

80 85

Ala Ala Pro Xaa Arg His Pro Ile Xaa Ile Lys Xaa Gly

90 95 100

Asp Trp Xaa Glu Phe Arg Xaa Lys Leu Thr Phe Tyr Leu

105 110

Xaa Thr Leu Glu Xaa Ala Gln Xaa Gln Gln

wherein m is 0 or 1; n is 0 or 1; p is 0 or 1; Xaa at position 4 is Asn or Ile; Xaa at position 11 is Thr or His; Xaa at position 15 is Gln, Arg, or Val; Xaa at position 18 is Leu, Ala, or Asn; Xaa at position 23 is Phe, Pro, or Ser; Xaa at position 28 is Glu, Ala, or Ser; Xaa at position 31 is Gln, Val, or Met; Xaa at position 37 is Asn or Arg; Xaa at position 41 is Arg, Leu, or Thr; Xaa at position 45 is Glu or Leu; Xaa at position 46 is Ala or Ser; Xaa at position 48 is Asn or Val; Xaa at position 53 is Ser, Asn, or His; Xaa at position 55 is Gln or Glu; Xaa at position 59 is Ala or Gly; Xaa at position 62 is Ser or Ala; Xaa at position 65 is Lys or Arg; Xaa at position 68 is Leu, Glu, or Val; Xaa at position 73 is Leu or Ser; Xaa at position 79 is Pro or Ser; Xaa at position 84 is His, Ile, or Thr; Xaa at position 87 is Asp or Ala; Xaa at position 91 is Asn or Glu; Xaa at position 95 is Arg or Glu; Xaa at position 102 is Lys or Val,; Xaa at position 106 is Asn, Gln, or His; Xaa at position 109 is Ala or Glu;

wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native (15-125)human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

The present invention includes polypeptides of Formula IX and Formula X above wherein from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3 or native (15-125) human interleukin-3; or a polypeptide having substantially the same structure and substantially the same biological activity.

“Mutant amino acid sequence,” “mutant protein” or “mutant polypeptide” refers to a polypeptide having an amino acid sequence which varies from a native sequence or is encoded by a nucleotide sequence intentionally made variant from a native sequence. “Mutant protein,” “variant protein” or “mutein” means a protein comprising a mutant amino acid sequence and includes polypeptides which differ from the amino acid sequence of native hIL-3 due to amino acid deletions, substitutions, or both. “Native sequence” refers to an amino acid or nucleic acid sequence which is identical to a wild-type or native form of a gene or protein.

Human IL-3 can be characterized by its ability to stimulate colony formation by human hematopoietic progenitor cells. The colonies formed include erythroid, granulocyte, megakaryocyte, granulocytic macrophages and mixtures thereof. Human IL-3 has demonstrated an ability to restore bone marrow function and peripheral blood cell populations to therapeutically beneficial levels in studies performed initially in primates and subsequently in humans (Gillio, A. P., et al. (1990); Ganser, A., et al. (1990); Falk, S., et al. (1991). Additional activities of hIL-3 include the ability to stimulate leukocyte migration and chemotaxis; the ability to prime human leukocytes to produce high levels of inflammatory mediators like leukotrienes and histamine; the ability to induce cell surface expression of molecules needed for leukocyte adhesion; and the ability to trigger dermal inflammatory responses and fever. Many or all of these biological activities of hIL-3 involve signal transduction and high affinity receptor binding. Mutant polypeptides of the present invention may exhibit useful properties such as having similar or greater biological activity when compared to native hIL-3 or by having improved half-life or decreased adverse side effects, or a combination of these properties. They may also be useful as antagonists. hIL-3 mutant polypeptides which have little or no activity when compared to native hIL-3 may still be useful as antagonists, as antigens for the production of antibodies for use in immunology or immunotherapy, as genetic probes or as intermediates used to construct other useful hIL-3 muteins. Since hIL-3 functions by binding to its receptor(s) and triggering second messages resulting in competent signal transduction, hIL-3 muteins of this invention may be useful in helping to determine which specific amino acid sequences are responsible for these activities.

The novel hIL-3 mutant polypeptides of the present invention will preferably have at least one biological property of human IL-3 or of an IL-3-like growth factor and may have more than one IL-3-like biological property, or an improved property, or a reduction in an undesirable biological property of human IL-3. Some mutant polypeptides of the present invention may also exhibit an improved side effect profile. For example, they may exhibit a decrease in leukotriene release or histamine release when compared to native hIL-3 or (15-125) hIL-3. Such hIL-3 or hIL-3-like biological properties may include one or more of the following biological characteristics and in vivo and in vitro activities.

One such property is the support of the growth and differentiation of progenitor cells committed to erythroid, lymphoid, and myeloid lineages. For example, in a standard human bone marrow assay, an IL-3-like biological property is the stimulation of granulocytic type colonies, megakaryocytic type colonies, monocyte/macrophage type colonies, and erythroid bursts. Other IL-3-like properties are the interaction with early multipotential stem cells, the sustaining of the growth of pluripotent precursor cells, the ability to stimulate chronic myelogenous leukemia (CML) cell proliferation, the stimulation of proliferation of mast cells, the ability to support the growth of various factor-dependent cell lines, and the ability to trigger immature bone marrow cell progenitors. Other biological properties of IL-3 have been disclosed in the art. Human IL-3 also has some biological activities which may in. some cases be undesirable, for example the ability to stimulate leukotriene release and the ability to stimulate increased histamine synthesis in spleen and bone marrow cultures and in vivo.

Biological activity of hIL-3 and hIL-3 mutant proteins of the present invention is determined by DNA synthesis by human acute myelogenous leukemia cells (AML). The factor-dependent cell line AML 193 was adapted for use in testing biological activity.

One object of the present invention is to provide hIL-3 muteins and hIL-3 deletion muteins with one or more amino acid substitutions in the polypeptide sequence which have similar of improved biological activity in relation to native hIL-3 or native (15-125) hIL-3.

The present invention includes mutant polypeptides comprising minimally amino acid residues 15 to 118 of hIL-3 with or without additional amino acid extensions to the N-terminus and/or C-terminus which further contain from one to three or more amino acid substitutions in the amino acid sequence of the polypeptide. It has been found that the (15-125) hIL-3 mutant is more soluble than is hIL-3 when expressed in the cytoplasm of

E. coli

, and the protein is secreted to the periplasm in

E. coli

at higher levels compared to native hIL-3.

When expressed in the

E. coli

cytoplasm, the above-mentioned mutant hIL-3 polypeptides of the present invention may also be constructed with Met-Ala- at the N-terminus so that upon expression the Met is cleaved off leaving Ala at the N-terminus. These mutant hIL-3 polypeptides may also be expressed in

E. coli

by fusing a signal peptide to the N-terminus. This signal peptide is cleaved from the polypeptide as part of the secretion process. Secretion in

E. coli

can be used to obtain the correct amino acid at the N-terminus (e.g., Asn

15

in the (15-125) hIL-3 polypeptide) due to the precise nature of the signal peptidase. This is in contrast to the heterogeneity often observed at the N-terminus of proteins expressed in the cytoplasm in

E. coli.

The hIL-3 mutant polypeptides of the present invention may have hIL-3 or hIL-3-like activity. For example, they may possess one or more of the biological activities of native hIL-3 and may be useful in stimulating the production of hematopoietic cells by human or primate progenitor cells. The hIL-3 muteins of the present invention and pharmaceutical compositions containing them may be useful in the treatment of conditions in which hematopoietic cell populations have been reduced or destroyed due to disease or to treatments such as radiation or chemotherapy.

hIL-3 muteins of the present invention may also be useful as antagonists which block the hIL-3 receptor by binding specifically to it and preventing binding of the agonist.

One potential advantage of the (15-125) hIL-3 muteins of the present invention, particularly those which retain activity similar to or better than that of native hIL-3, is that it may be possible to use a smaller amount of the biologically active mutein to produce the desired therapeutic effect. This may make it possible to reduce the number of treatments necessary to produce the desired therapeutic effect. The use of smaller amounts may also reduce the possibility of any potential antigenic effects or other possible undesirable side effects. For example, if a desired therapeutic effect can be achieved with a smaller amount of polypeptide it may be possible to reduce or eliminate side effects associated with the administration of native IL-3 such as the stimulation of leukotriene and/or histamine release. The hIL-3 muteins of the present invention may also be useful in the activation of stem cells or progenitors which have low receptor numbers. Pharmaceutical compositions containing hIL-3 muteins of the present invention can be administered parenterally, intravenously, or subcutaneously.

In variants which contain an additional cysteine the presence of the cysteine permits the labeling of the protein with ricin which permits targeting ricin and other toxins or tracers using a sulfhydryl linkage to the hIL-3 receptor.

As another aspect of the present invention, there is provided a novel method for producing the novel family of human IL-3 muteins. The method of the present invention involves culturing a suitable cell or cell line( which has been transformed with a vector containing a DNA sequence coding for expression of a novel hIL-3 mutant polypeptide. Suitable cells or cell lines may be bacterial cells. For example, the various strains of

E. coli

are well-known as host cells in the field of biotechnology. Examples of such strains include

E. coli

strains JM101 [Yanish-Perron, et al. (1985)] and MON105 [Obukowicz, et al. (1992)]. Various strains of

B subtilis

may also be employed in this method. Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention.

Also suitable for use in the present invention are mammalian cells, such as Chinese hamster ovary cells (CHO). General methods for expression of foreign genes in mammalian cells are reviewed in; Kaufman, R. J. (1987) High level production of proteins-in mammalian cells, in

Genetic Engineering, Principles and Methods

, Vol. 9, J. K. Setlow, editor, Plenum Press, New York. An expression vector is constructed in which a strong promoter capable of functioning in mammalian cells drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally fused to the coding region for the hIL-3 variant. For example, plasmids such as pcDNA I/Neo, pRc/RSV, and pRc/CMV (obtained from Invitrogen Corp., San Diego, Calif.) can be used. The eukaryotic secretion signal peptide coding region can be from the hIL-3 gene itself or it can be from another secreted mammalian protein (Bayne, M. L. et al. (1987)

Proc. Natl. Acad. Sci. USA

84, 2638-2642). After construction of the vector containing the hIL-3 variant gene, the vector DNA is transfected into mammalian cells. Such cells can be, for example, the COS7, HeLa, BHK, CHO, or mouse L lines. The cells can be cultured, for example, in DMEM media (JRH Scientific). The hIL-3 variant secreted into the media can be recovered by standard biochemical approaches following transient expression 24-72 hours after transfection of the cells or after establishment of stable cell lines following selection for neomycin resistance. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Gething and Sambrook,

Nature,

2:620-625 (1981), or alternatively, Kaufman et al,

Mol. Cell. Biol.

, 5(7):1750-1759 (1985) or Howley et al., U.S. Pat. No. 4,419,446. Another suitable mammalian cell line is the monkey COS-1 cell line. A similarly useful mammalian cell line is the CV-1 cell line.

Where desired, insect cells may be utilized as host cells in the method of the present invention. See, e.g. Miller et al,

Genetic Engineering,

8:277-298 (Plenum Press 1986) and references cited therein. In addition, general methods for expression of-foreign genes in insect cells using Baculovirus vectors are described in: Summers, M. D. and Smith, G. E. (1987)—A manual of methods for Baculovirus vectors and insect cell culture procedures, Texas Agricultural Experiment Station Bulletin No. 1555. An expression vector is constructed comprising a Baculovirus transfer vector, in which a strong Baculovirus promoter (such as the polyhedron promoter) drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally fused to the coding region for the hIL-3 variant polypeptide. For example, the plasmid pVL1392 (obtained from Invitrogen Corp., San Diego, Calif.) can be used. After construction of the vector carrying the hIL-3 variant gene, two micrograms of this DNA is cotransfected with one microgram of Baculovirus DNA (see Summers & Smith, 1987) into insect cells, strain SF9. Pure recombinant Baculovirus carrying the hIL-3 variant is used to infect cells cultured, for example, in Excell 401 serum-free medium (in Biosciences, Lenexa, Kans.). The hIL-3 variant secreted into the medium can be recovered by standard biochemical approaches.

Another aspect of the present invention provides plasmid DNA vectors for use in the method of expression of these novel hIL-3 muteins. These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention. Appropriate vectors which can transform microorganisms capable of expressing the hIL-3 muteins include expression vectors comprising nucleotide sequences coding for the hIL-3 muteins joined to transcriptional and translational regulatory sequences which are selected according to the host cells used.

Vectors incorporating modified sequences as described above are included in the present invention and are useful in the production of the hIL-3 mutant polypeptides. The vector employed in the method also contains selected regulatory sequences in operative association with the DNA coding sequences of the invention and capable of directing the replication and expression thereof in selected host cells.

The present invention also includes the construction and expression of (15-125)human interleukin-3 muteins having one or more amino acid substitutions in secretion vectors that optimize accumulation of correctly folded, active polypeptide. While many heterologous proteins have been secreted in

E. coil

there is still a great deal of unpredictability and limited success (Stader and Silhavy 1990). Full-length hIL-3 is such a protein, where attempts to secrete the protein in

E. coli

resulted in low levels of secretion. Secretion of the variant (15-125) hIL-3 mutant polypeptides of the present invention as a fusion with a signal peptide such as lamB results in correctly folded protein that can be removed from the periplasm of

E. coli

by osmotic shock fractionation. This property of the variant (15-125) hIL-3 muteins allows for the direct and rapid screening for bioactivity of the secreted material in the crude osmotic shock fraction, which is a significant advantage. Furthermore, it provides a means of using the (15-125) hIL-3 muteins to conduct structure activity relationship (SAR) studies of the hIL-3 molecule. A further advantage of secretion of (15-125) hIL-3 muteins fused to the lamb signal peptide is that the secreted polypeptide has the correct N-terminal amino acid (Asn) due to the precise nature of the cleavage of the signal peptide by signal peptidase, as part of the secretion process.

The (15-125) hIL-3 muteins of the present invention may include hIL-3 polypeptides having Met-, Ala- or Met-Ala- attached to the N-terminus. When the muteins are expressed in

E. coli

, polypeptides with and without Met attached to the N-terminus are obtained. The methlonine can in some cases be removed by methionine aminopeptidase.

Amino terminal sequences of some of the hIL-3 muteins made in

E. coli

were determined using the method described by Hunkapillar et al., (1983). It was found that hIL-3 proteins made in

E. coli

from genes encoding Met-(15-125) hIL-3 were isolated as Met-(15-125) hIL-3. Proteins produced from genes encoding Met-Ala-(15-125) hIL-3 were produced as Ala-(15-125) hIL-3. The N-termini of proteins made in the cytoplasm of

E. coli

are affected by posttranslational processing by methionine aminopeptidase (Ben-Bassat et al., 1987) and possibly by other peptidases.

One method of creating the preferred hIL-3 (15-125) mutant genes is cassette mutagenesis [Wells, et al. (1985)] in which a portion of the coding sequence of hIL-3 in a plasmid is replaced with synthetic oligonucleotides that encode the desired amino acid substitutions in a portion of the gene between two restriction sites. In a similar manner amino acid substitutions could be made in the full-length hIL-3 gene, or genes encoding variants of hIL-3 in which from 1 to 14 amino acids have been deleted from the N-terminus and/or from 1 to 15 amino acids have been deleted from the C-terminus. When properly assembled these oligonucleotides would encode hIL-3 variants with the desired amino acid substitutions and/or deletions from the N-terminus and/or C-terminus. These and other mutations could be created by those skilled in the art by other mutagenesis methods including; oligonucleotide-directed mutagenesis [Zoller and Smith (1982, 1983, 1984), Smith (1985), Kunkel (1985), Taylor, et al. (1985), Deng and Nickoloff (1992)] or polymerase chain reaction (PCR) techniques [Saiki, (1985)].

Pairs of complementary synthetic oligonucleotides encoding portions of the amino terminus of the hIL-3 gene can be made and annealed to each other. Such pairs would have protruding ends compatible with ligation to NcoI at one end. The NcoI site would include the codon for the initiator methionine. At the other end of oligonucleotide pairs, the protruding (or blunt) ends would be compatible with a restriction site that occurs within the coding sequence of the hIL-3 gene. The DNA sequence of the oligonucleotide would encode sequence for amino acids of hIL-3 with the exception of those substituted and/or deleted from the sequence.

The NcoI enzyme and the other restriction enzymes chosen should have recognition sites that occur only once in the DNA of the plasmid chosen. Plasmid DNA can be treated with the chosen restriction endonucleases then ligated to the annealed oligonucleotides. The ligated mixtures can be used to transform competent JM101 cells to resistance to an appropriate antibiotic. Single colonies can be picked and the plasmid DNA examined by restriction analysis and/or DNA sequencing to identify plasmids with mutant hIL-3 genes.

One example of a restriction enzyme which cleaves within the coding sequence of the hIL-3 gene is ClaI whose recognition site is at codons 20 and 21. The use of ClaI to cleave the sequence of hIL-3 requires that the plasmid DNA be isolated from an

E. coli

strain that fails to methylate adenines in the DNA at GATC recognition sites. This is because the recognition site for ClaI, ATCGAT, occurs within the sequence GATCGAT which occurs at codons 19, 20 and 21 in the hIL-3 gene. The A in the GATC sequence is methylated in most

E. coli

host cells. This methylation prevents ClaI from cleaving at that particular sequence. An example of a strain that does not methylate adenines is GM48.

Interpretation of Activity of Single Amino Acid Mutants in IL-3 (15-125)

As illustrated in Tables 6 and 9, there are certain positions in the IL-3 (15-125) molecule which are intolerant of substitutions, in that most or all substitutions at these positions resulted in a considerable decrease in bioactivity. There are two likely classes of such “down-mutations”: mutations that affect overall protein structure, and mutations that interfere directly with the interaction between the IL-3 molecule and its receptor. Mutations affecting the three-dimensional structure of the protein will generally lie in the interior of the protein, while mutations affecting receptor binding will generally lie on the surface of the protein. Although the three-dimensional structure of IL-3 is unknown, there are simple algorithms which can aid in the prediction of the structure. One such algorithm is the use of “helical wheels” (Kaiser, E. T. & Kezdy, F. J., Science, 223:249-255 (1984)). In this method, the presence of alpha helical protein structures can be predicted by virtue of their amphipathic nature. Helices in globular proteins commonly have an exposed hydrophilic side and a buried hydrophobic side. As a broad generalization, in globular proteins, hydrophobic residues are present in the interior of the protein, and hydrophilic residues are present on the surface. By displaying the amino acid sequence of a protein on such a “helical wheel” it is possible to derive a model for which amino acids in alpha helices are exposed and which are buried in the core of the protein. Such an analysis of the IL-3 (15-125) molecule predicts that the following helical residues are buried in the core:

M19, I20, I23, I24, L27, L58, F61, A64, L68, A71, I74, I77, L78, L81, W104, F107, L111, Y114, L115, L118.

In addition, cysteine residues at positions 16 and 84 are linked by a disulfide bond, which is important for the overall structure or “folding” of the protein. Finally, mutations which result in a major disruption of the protein structure may be expressed at low level in the secretion system used in our study, for a variety of reasons: either because the mis-folded protein is poorly recognized by the secretion machinery of the cell; because mis-folding of the protein results in aggregation, and hence the protein cannot be readily extracted from the cells; or because the mis-folded protein is more susceptible to degradation by cellular proteases. Hence, a block in secretion may indicate which positions in the IL-3 molecule which are important for maintenance of correct protein structure.

In order to retain the activity of a variant of IL-3, it is necessary to retain both the structural integrity of the protein, and retain the specific residues important for receptor contact. Hence it is possible to define specific amino acid residues in IL-3 (15-125) which must be retained in order to preserve biological activity.

Residues predicted to be important for interaction with the receptor: D21, E22, E43, D44, L48, R54, R94, D103, K110, F113.

Residues predicted to be structurally important: C16, L58, F61, A64, I74, L78, L81, C84, P86, P92, P96, F107, L111, L115, L118.

The hIL-3 muteins of the present invention may be useful in the treatment of diseases characterized by a decreased levels of either myeloid, erythroid, lymphoid, or megakaryocyte cells of the hematopoietic system or combinations thereof. In addition, they may be used to activate mature myeloid and/or lymphoid cells. Among conditions susceptible to treatment with the polypeptides of the present invention is leukopenia, a reduction in the number of circulating leukocytes (white cells) in the peripheral blood. Leukopenia may be induced by exposure to certain viruses or to radiation. It is often a side effect of various forms of cancer therapy, e.g., exposure to chemotherapeutic drugs and of infection or hemorrhage. Therapeutic treatment of leukopenia with these hIL-3 mutant polypeptides of the present invention may avoid undesirable side effects caused by treatment with presently available drugs.

The hIL-3 muteins of the present invention may be useful in the treatment of neutropenia and, for example, in the treatment of such conditions as aplastic anemia, cyclic neutropenia, idiopathic neutropenia, Chdiak-Higashi syndrome, systemic lupus erythematosus (SLE), leukemia, myelodysplastic syndrome and myelofibrosis.

Many drugs may cause bone marrow suppression or hematopoietic deficiencies. Examples of such drugs are AZT, DDI, alkylating agents and anti-metabolites used in chemotherapy, antibiotics such as chloramphenicol, penicillin and sulfa drugs, phenothiazones, tranquilizers such as meprobamate, and diuretics. The hIL-3 muteins of the present invention may be useful in preventing or treating the bone marrow Suppression or hematopojetic deficiencies which often occur in patients treated with these drugs.

Hematopoietic deficiencies may also occur as a result of viral, microbial or parasitic infections and as a result of treatment for renal disease or renal failure, e.g., dialysis. The hIL-3 muteins of the present invention may be useful in treating such hematopoietic deficiency.

The treatment of hematopoietic deficiency may include administration of the hIL-3 mutein of a pharmaceutical composition containing the hIL-3 mutein to a patient. The hIL-3 muteins of the present invention may also be useful for the activation and amplification of hematopoietic precursor cells by treating these cells in vitro with the muteins of the present invention prior to injecting the cells into a patient.

Various immunodeficiencies e.g., in T and/or B lymphocytes, or immune disorders, e.g., rheumatoid arthritis, may also be beneficially affected by treatment with the hIL-3 mutant polypeptides of the present invention. Immunodeficiencies may be the result of viral infections e.g. HTLVI, HTLVII, HTLVIII, severe exposure to radiation, cancer therapy or the result of other medical treatment. The hIL-3 mutant polypeptides of the present invention may also be employed, alone or in combination with other hematopoietins, in the treatment of other blood cell deficiencies, including thrombocytopenia (platelet deficiency), or anemia. Other uses for these novel polypeptides are in the treatment of patients recovering from bone marrow transplants in vivo and ex vivo, and in the development of monoclonal and polyclonal antibodies generated by standard methods for diagnostic or therapeutic use.

Other aspects of the present invention are methods and therapeutic compositions for treating the conditions referred to above. Such compositions comprise a therapeutically effective amount of one or more of the hIL-3 muteins of the present invention in a mixture with a pharmaceutically acceptable carrier. This composition can be administered either parenterally, intravenously or subcutaneously. When administered, the therapeutic composition for use in this invention is preferably in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such a parenterally acceptable protein solution, having due regard to pH, isotonicity, stability and the like, is within the skill of the art.

The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician considering various factors which modify the action of drugs, e.g. the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, a daily regimen may be in the range of 0.2-150 μg/kg of non-glycosylated IL-3 protein per kilogram of body weight. This dosage regimen is referenced to a standard level of biological activity which recognizes that native IL-3 generally possesses an EC

50

at or about 10 picoMolar to 100 picoMolar in the AML proliferation assay described herein. Therefore, dosages would be adjusted relative to the activity of a given mutein vs. the activity of native (reference) IL-3 and it would not be unreasonable to note that dosage regimens may include doses as low as 0.1 microgram and as high as 1 milligram per kilogram of body weight per day. In addition, there may exist specific circumstances where dosages of IL-3 mutein would be adjusted higher or lower than the range of 10-200 micrograms per kilogram of body weight. These include co-administration with other CSF or growth factors; co-administration with chemotherapeutic drugs and/or radiation; the use of glycosylated IL-3 mutein; and various patient-related issues mentioned earlier in this section. As indicated above, the therapeutic method and compositions may also include co-administration with other human factors. A non-exclusive list of other appropriate hematopoietins, CSFs and interleukins for simultaneous or serial co-administration with the polypeptides of the present invention includes GM-CSF, CSF-1, G-CSF, Meg-CSF, M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, LIF, B-cell growth factor, B-cell differentiation factor and eosinophil differentiation factor, stem cell factor (SCF) also known as steel factor or c-kit ligand, or combinations thereof. The dosage recited above would be adjusted to compensate for such additional components in the therapeutic composition. Progress of the treated patient can be monitored by periodic assessment of the hematological profile, e.g., differential cell count and the like.

Materials and Methods for hTL-1 Mutein Expression in

E. coli

Unless noted otherwise, all specialty chemicals were obtained from Sigma Co., (St. Louis, Mo.). Restriction endonucleases, T4 poly-nucleotides kinase,

E. coli

DNA polymerase I large fragment (Klenow) and T4 DNA ligase were obtained from New England Biolabs (Beverly, Mass.) or Boehringer Mannheim (Indianapolis, Ind.). All chemicals and enzymes were used according to manufacturer's directions.

Escherichia coli

Strains

Strain JM101: delta (pro lac), supE, thi, F′ (traD36, proAB, lacI-Q, lacZdeltaM15) (Messing, 1979). This strain can be obtained from the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, accession number 33876. MON 105 (W3110 rpoH358) (Obukowicz, et al., 1992) is a derivative of W3110 (Bachmann, 1972) and has been assigned ATCC accession number 55204. Strain GM48: dam-3, dcm-6, gal, ara, lac, thr, leu, tonA, tsx (Marinus, 1973) was used to make plasmid DNA that is not methylated at the sequence GATC.

Genes and Plasmids

The gene used for hIL-3 production in

E. coli

was obtained from British Biotechnology Incorporated, Cambridge, England, catalogue number BBG14. This gene is carried on a pUC based plasmid designated pP0518. The human IL-3 gene sequence is from Yang, et al. (1986).

The plasmids used for production of hIL-3 in

E. coli

contain genetic elements whose use has been described (Olins et al., 1988; Olins and Rangwala, 1990). The replicon used is that of pBR327 [(Bolivar et al. (1977); Soberon et al., 1980] which is maintained at a copy number of about 50 in the cell (Covarrubias, et al., (1981)). A gene encoding the beta-lactamase protein is present on the plasmids. This protein confers ampicillin resistance on the cell. This resistance serves as a selectable phenotype for the presence of the plasmid in the cell.

Intracellular Expression Plasmids

For cytoplasmic (intracellular) expression vectors the transcription promoter was derived from the recA gene of

E. coli

(Sancar et al., 1980). This promoter, designated precA, is contained on 72 base pairs (bp) BglII, BamHI fragment which includes the RNA polymerase binding site and the lexA repressor binding site (the operator). This segment of DNA provides high level transcription that is regulated even when the recA promoter is on a plasmid with the pBR327 origin of replication (Olins et al., 1988) incorporated herein by reference.

Secretion Expression Plasmids

In secretion expression plasmids the transcription promoter was derived from the ara B, A. and D genes of

E. coli

(Greenfield et al., 1978). This promoter is designated pAraBAD and is contained on a 323 base pair SacrII, BglII restriction fragment. The lamB secretion leader (Wong et al., 1988, Clement et al., 1981) was fused to the N-terminus of the hIL-3 gene at the recognition sequence for the enzyme NcoI (5′CCATGG3′). The hIL-3 genes used were engineered to have a HindIII recognition site (5′AAGCTT3′) following the coding sequence of the gene. Downstream of the gene is a 550 bp fragment containing the origin of replication of the single stranded phage f1 [Olins and Rangwala (1989)].

These hIL-3 variants were expressed as a fusion with the lamB signal peptide operatively joined to the araBAD promoter (Greenfield, 1978) and the g10-L ribosome binding site (Olins et al. 1988). The signal peptide is removed as part of the secretion process. The processed form was selectively released from the periplasm by osmotic shock as a correctly folded and fully active molecule. Secretion of (15-125) hIL-3 was further optimized by using low inducer (arabinose) concentration and by growth at 30° C. These conditions resulted in lower accumulation levels of unprocessed lamB signal peptide (15-125) hIL-3 fusion, maximal accumulation levels of processed (15-125) hIL-3 and selective release of (15-125) hIL-3 by osmotic shock fractionation. The use of a tightly regulated promoter such as araBAD from which the transcription level and hence the expression level can be modulated allowed for the optimization of secretion of (15-125) hIL-3.

The ribosome binding site (RBS) used is that from gene 10 of phage T7 (Olins et al., 1988). This is encoded in a 100 base pair (bp) fragment placed adjacent to precA. In the plasmids used herein, the recognition sequence for the enzyme NcoI (5′CCATGG3′) follows the g10-L RBS. It is at this NcoI site that the hIL-3 genes are joined to the plasmid. It is expected that the nucleotide sequence at this junction will be recognized in mRNA as a functional start site for translation (Olins et al., 1988). The hIL-3 genes used were engineered to have a HindIII recognition site (5′AAGCTT3′) following the coding sequence of the gene. Downstream of the gene is a 550 base pair fragment containing the origin of replication of the single stranded phage f1 (Dente et al., 1983; Olins, et al., 1990) both incorporated herein by reference. A plasmid containing these elements is pMON2341. Another plasmid containing these elements is pMON5847 which has been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 under the accession number ATCC 68912.

Synthesis of Oligonucleotides

oligonucleotides were synthesized by the cyanoethyl method (Addam et al. 1983, McBride, et al. 1983, Sinba et al., 1984) on Nucleotide Synthesizer model 380A or 380B from Applied Biosystems, Inc. (Foster City, Calif.). Some oligonucleotides were purchased from Genosys Biotechnologies Inc. (The Woodlands, Tex.) or Midland Certified Reagent Co. (Midland, Tex.). The degenerate oligonucleotides were synthesized by machine mixing an equal molar ratio of the desired nucleosides in the condensation reaction at degenerate positions. Oligonucleotides were purified by polyacrylamide gel electrophoresis at concentrations from 12-20% (19:1 crosslinked) in 0.5×Tris borate (TBE) buffer (0.045 M Tris, 0.045 M boric acid, 1.25 mM EDTA) as described by Atkinson (1984) . The Oligonucleotides were desalted by passage through a Nensorb 20 column obtained from DuPont/New England Nuclear (Boston, Mass.) using a PREP Automated Sample Processor obtained from DuPont, Co. (Wilmington, Del.).

Quantitation of Synthetic Oligonucleotides

Synthetic oligonucleotides were resuspended in water (100 μl) and quantitated by reading the absorbance at 260 nm on a Beckman DU40 Spectrophotometer (Irvine, Calif.) using a one centimeter by one millimeter quartz cuvette (Maniatis, 1982). The concentration was determined using an extinction coefficient of 1×10

4

(Voet et al., 1963; Mahler and Cordes, 1966). The oligonucleotide was then diluted to the desired concentration.

Quantitation of synthetic DNA fragments can also be achieved by adding 10 to 100 picomoles of DNA to a solution containing kinase buffer (25 mM Tris pH 8.0, 10 mM MgCl

2

, 10 mM DTT and 2 mM spermidine). To the reaction mix is added ATP to 20 micromolar, ATP radiolabeled at the gamma phosphate (5000-10,0000 dpm/pmol) and 5 units of T4 polynucleotide kinase. Radiolabelled material is obtained from New England Nuclear (Boston, Mass.). The 10 microliter mixture is incubated at 37° C. for one hour. A 1 microliter aliquot of the mixture is chromatographed on DEAE paper (DE81 from Whatman) in 0.35 M ammonium bicarbonate. The counts that remain at the origin are used to determine the concentration of the synthetic DNA.

Recombinant DNA Methods

Isolation of plasmid DNA from

E. coli

cultures was performed as described (Birnboim and Doly, 1979). Some DNAs were purified by Magic™ miniprep columns, available from Promega (Madison, Wis.).

Purified plasmid DNA was treated with restriction endonucleases according to manufacturer's instructions. Analysis of the DNA fragments produced by treatment with restriction enzymes was done by agarose or polyacrylamide gel electrophoresis. Agarose (DNA grade from Fisher, Pittsburgh Pa.) was used at a concentration of 1.0% in a Tris-acetate running buffer (0.04 M Tris-acetate, 0.001M EDTA). Polyacrylamide (BioRad, Richmond Calif.) was used at a concentration of 6% (19:1 crosslinked) in 0.5×Tris-borate buffer (0.045 M Tris, 0.045 M boric acid, 1.25 mM EDTA), hereafter referred to as PAGE.

DNA polymerase I, large fragment, Klenow enzyme was used according to manufacturer's instructions to catalyze the addition of mononucleotides from 5′ to 3′ of DNA fragments which had been treated with restriction enzymes that leave protruding ends. The reactions were incubated at 65° C. for 10 minutes to heat inactivate the Klenow enzyme.

The synthetic oligonucleotides were made without 5′ or 3′ terminal phosphates. In cases where such oligonucleotides were ligated end to end, the oligonucleotides were treated at a concentration of 10 picomoles per microliter with T4 polynucleotide kinase in the following buffer: 25 mM Tris, pH 8.0, 10 mM MgCl

2

, 10 mM dithiothreitol, 2 mM spermidine, 1 mM rATP. After incubation for 30 minutes at 37° C., the samples were incubated at 65° C. for five minutes to heat inactivate the kinase.

Synthetic Gene Assembly

The (15-125) hIL-3 gene was divided into four regions separated by five convenient restriction sites. In each of the four regions synthetic oligonucleotides were designed so that they would anneal in complementary pairs, with protruding single stranded ends “or blunt ends” and when the pairs were properly assembled would result in a DNA sequence that encoded a portion of the hIL-3 gene. Amino acid substitutions in the hIL-3 gene were made by designing the oligonucleotides to encode the desired substitutions. The complementary oligonucleotides were annealed at concentration of 1 picomole per microliter in ligation buffer plus 50 mM NaCl. The samples were heated in a 100 ml beaker of boiling water and permitted to cool slowly to room temperature. One picomole of each of the annealed pairs of oligonucleotides were ligated with approximately 0.2 picomoles of plasmid DNA, digested with the appropriate restriction enzymes, in ligation buffer (25 mM Tris pH 8.0, 10 mM MgCl

2

, 10 mM dithiothreitol, 1 mM ATP, 2 mM spermidine) with T4 DNA ligase obtained from New England Biolabs (Beverly, Mass.) in a total volume of 20 μl at room temperature overnight.

DNA fragments were isolated from agarose gels by intercepting the restriction fragments on DEAE membranes from Schleicher and Schuell (Keene, N.H.) and eluting the DNA in 10 mM Tris, 1 mM EDTA, 1 M NaCl at 55° C. for 1 hour, according to manufacturer's directions. The solutions containing the DNA fragment were concentrated and desalted by using Centricon 30 concentrators from Amicon (W. R. Grace, Beverly Mass.) according to the manufacturer's directions. Ligations were performed at 15° C. overnight, except as noted, in ligation buffer (66 mM Tris pH 7.5, 6.6 mM MgCl

2

, 1 mM dithiothreitol, 0.4 mM ATP) with T

4

ligase obtained from New England Biolabs (Beverly, Mass.).

Polymerase Chain Reaction

Polymerase Chain Reaction (hereafter referred to as PCR) techniques (Saiki, 1985) used the reagent kit and thermal cycler from Perkin-Elmer Cetus (Norwalk, Conn.). PCR is based on a thermostable DNA polymerase from

Thermus aquaticus

. The PCR technique is a DNA amplification method that mimics the natural DNA replication process in that the number of DNA molecules doubles after each cycle, in a way similar to in vivo replication. The DNA polymerase mediated extension is in a 5′→3′ direction. The term “primer” as used herein refers to an oligonucleotide sequence that provides an end to which the DNA polymerase can add nucleotides that are complementary to a nucleotide sequence. The latter nucleotide sequence is referred to as the “template”, to which the primers are annealed. The amplified PCR product is defined as the region comprised between the 5′ ends of the extension primers. Since the primers have defined sequences, the product will have discrete ends, corresponding to the primer sequenCes. The primer extension reaction was carried out using 20 picomoles (pmoles) of each of the oligonucleotides and 1 picogram of template plasmid DNA for 35 cycles (1 cycle is defined as 94° C. for one minute, 50° C. for two minutes and 72° C. for three minutes). The reaction mixture was extracted with an equal volume of phenol/chloroform (50% phenol and 50% chloroform, volume to volume) to remove proteins. The aqueous phase, containing the amplified DNA, and solvent phase were separated by centrifugation for 5 minutes in a microcentrifuge (Model 5414 Eppendorf Inc, Fremont Calif.). To precipitate the amplified DNA the aqueous phase was removed and transferred to a fresh tube to which was added 1/10 volume of 3M NaOAc (pH 5.2) and 2.5 volumes of ethanol (100% stored at minus 20° C.). The solution was mixed and placed on dry ice for 20 minutes. The DNA was pelleted by centrifugation for 10 minutes in a microcentrifuge and the solution was removed from the pellet. The DNA pellet was washed with 70% ethanol, ethanol removed and dried in a speedvac concentrator (Savant, Farmingdale, N.Y.). The pellet was resuspended in 25 microliters of TE (20 mM Tris-HCl pH 7.9, 1 mM EDTA). Alternatively the DNA was precipitated by adding equal volume of 4M NH

4

OAc and one volume of isopropanol [Treco, (1989)]. The solution was mixed and incubated at room temperature for 10 minutes and centrifuged. These conditions selectively precipitate DNA fragments larger than ˜20 bases and were used to remove oligonucleotide primers. One quarter of the reaction was digested with restriction enzymes [Higuchi, (1989)] and on completion heated to 70° C. to inactivate the enzymes.

Two Step Site-directed PCR Mutagenesis

Single amino acid substitution variants were created at positions 17-123 of hIL-3 in two site-directed mutagenesis steps by PCR (Bauer et al. manuscript in preparation).

The single amino acid substitution variants at positions 94-105 of hIL-3 were created as described below. In the first mutagenesis step plasmid DNA, containing the hIL-3 gene (amino acids 15-125), was the template in the PCR reaction. The DNA sequence of one of the oligonucleotide primers was designed to replace 12 base in the hIL-3 gene (15-125) with 12 bases encoding two translation stop codons (5′TAATAA3′), followed. by the recognition sequence (5′GTCGAC3′) restriction enzyme SalI. This 12 base sequence was substituted in the hIL-3 gene following the codon for amino acids 93, 97 and 101. Plasmids containing these mutagenized genes served as the templates for the second mutagenesis step.

In the second mutagenesis step, the 12 base substitution introduced in the first mutagenesis step, was replaced using a 32 fold degenerate oligonucleotide. The degenerate oligonucleotides were synthesized by machine mixing an equal molar ratio of the desired nucleosides in the condensation reaction at degenerate positions. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon. The other bases in the oligonucleotides corresponded to the hIL-3 sequence. The degenerate oligonucleotides theoretically contain 32 different codons, encoding all 20 amino acids and one translation stop codon, at a single position. At the other 9 bases the DNA sequence was restored to encode the native hIL-3 protein sequence. This pool of single amino acid substitutions at a single position is referred to as a “library”. This two step PCR site-directed mutagenesis approach was used to facilitate the identification of single amino acid substitution variants by differential DNA hybridization.

The single amino acid substitution variants at positions 17-93 and 106-123 of hIL-3 (15-125) were created as described below. In the first mutagenesis step plasmid DNA, containing the hIL-3 gene (15-125), was the template in the PCR reaction. The DNA sequence of one of the oligonucleotide primers was designed to delete 18 bases in the hIL-3 gene that encode the following amino acids; 17-22, 23-28, 29-34, 35-40, 41-46, 47-52, 53-58, 59-64, 65-70, 71-76, 77-82, 83-88, 88-93, 106-111, 112-117 and 118-123. Plasmids containing these deletion genes served as the templates for the second mutagenesis step.

In the second mutagenesis step the 18 base deletion, created in the first mutagenesis step, was restored using a 32 fold degenerate oligonucleotide. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon. The other bases in the oligonucleotides corresponded to the hIL-3 sequence. The degenerate oligonucleotides theoretically contain 32 different codons, encoding all 20 amino acids and one translation stop codon, at a single position. At the other 9 bases the DNA sequence was restored to encode the native hIL-3 protein sequence. This pool of single amino acid substitutions at a single position is referred to as a “library”. This two step PCR site-directed mutagenesis approach was used to facilitate the identification of single amino acid substitution variants by differential DNA hybridization.

Recovery of Recombinant Plasmids from Ligation Mixes and Transformation of

E. coli

Cells with Recombinant Plasmid DNA

E. coli

JM101 cells were made competent to take up DNA. Typically, 20 to 100 ml of cells were grown in LB medium to a density of approximately 150 Klett units and then collected by centrifugation. The cells were resuspended in one half culture volume of 50 mM CaCl

2

and held at 4° C. for one hour. The cells were again collected by centrifugation and resuspended in one tenth culture volume of 50 mM CaCl

2

. DNA was added to a 150 microliter volume of these cells, and the samples were held at 4° C. for 30 minutes. The samples were shifted to 42° C. for one minute, one milliliter of LB was added, and the samples were shaken at 37° C. for one hour. Cells from these samples were spread on plates containing ampicillin to select for transformants. The plates were incubated overnight at 37° C. Single colonies were picked and grown in LB supplemented with ampicillin overnight at 37° C. with shaking. From these cultures DNA was isolated for restriction analysis.

Typically plasmids were constructed, using methods described herein or by references cited herein, as follows except as noted in examples included herein. DNA fragments were purified from agarose or polyacrylamide gels. Purified DNA fragments were ligated and the ligation reaction mixture was used to transform

E. coli

K-12 strain JM101. Transformant bacteria were selected on ampicillin containing plates. Plasmid DNA was isolated from a single colony grown in LB Broth and screened by restriction analysis for the desired construct and sequenced to determine that the DNA sequence was correct.

Culture Media

LB medium (Maniatis et al., 1982) was used for growth of cells for DNA isolation. M9 minimal medium supplemented with 1.0% casamino acids, acid hydrolyzed casein, Difco (Detroit, Mich.) was used for cultures in which recombinant hIL-3 was produced. The ingredients in the M9 medium were as follows: 3g/liter KH

2

PO

4

, 6 g/l Na

2

HPO

4

, 0.5 g/l NaCl, 1 g/l NH

4

Cl, 1.2 mM MgSO

4

, 0.025 mM CaCl

2

, 0.2% glucose (0.2% glycerol with the AraBAD promoter), 1% casamino acids, 0.1 ml/l trace minerals (per liter 108 g FeCl

3

. 6H

2

O, 4.0 g ZnSO

4

.7H

2

O, 7.0 CoCl

2

.2H

2

O, 7.0 g Na

2

MoO

4

.2H

2

O, 8.0 g CuSO

4

.5H

2

O, 2.0 g H

3

BO

3

, 5.0 g MnSO

4

.H

2

O, 100 ml concentrated HCl). Bacto agar from Difco was used for solid media and ampicillin (Polycillin-N from Bristol-Meyers, Evansville, Ind.) was added to both liquid and solid LB media at 200 micrograms per milliliter.

DNA Sequence Analysis

The nucleotide sequencing of plasmid DNA was performed using a Genesis 2000 sequencer obtained from DuPont (Wilmington, Del.) according to the methods of Prober et al. (1987) and Sanger et al. (1977). Some DNA sequences were determined using Sequenase™ polymerase according to the protocol of its supplier, U.S. Biochemicals (Cleveland, Ohio).

Production of Recombinant hIL-3 Muteins in

E. coli

with Vectors Employing the recA Promoter

E. coli

strains harboring the plasmids of interest were grown at 37° C. in M9 plus casamino acids medium with shaking in a Gyrotory water bath Model G76 from New Brunswick Scientific (Edison, N.J.). Growth was monitored with a Klett Summerson meter (green 54 filter), Klett Mfg. Co. (New York, N.Y.). At a Klett value of approximately 150, an aliquot of the culture (usually one milliliter) was removed for protein analysis. To the remaining culture, nalidixic acid (10 mg/ml) in 0.1 N NaOH was added to a final concentration of 50 μg/ml. The cultures were shaken at 37° C. for three to four hours after addition of nalidixic acid. A high degree of aeration was maintained throughout the bacterial growth in order to achieve maximal production of the desired gene product. The cells were examined under a light microscope for the presence of retractile bodies (RBs). One milliliter aliquots of the culture were removed for analysis of protein content.

Production of Recombinant hITL-3 Proteins from the AraBAD Promoter in

E. coli

E. coli

strains harboring the plasmids of interest were grown at 30° C. with shaking in M9 medium plus casamino acids and glycerol. Growth was monitored with a Klett Summerson calorimeter, using a green 54 filter. At a Klett value of about 150, an aliquot of the culture (usually one milliliter) was removed for protein analysis. To the remaining culture, 20% arabinose was added to a final concentration of 0.05%. The cultures were shaken at 30° C. for three to four hours after addition of arabinose. A high degree of aeration was maintained throughout the bacterial growth in order to achieve maximal production of the desired gene product. One milliliter aliquots of the culture were removed for analysis of protein content.

Secretion and Osmotic Shock

Three hour post induction samples were fractionated by osmotic shock [Neu and Heppel (1965)]. The Klett value of the cultures was determined and 1 ml of cells were centrifuged in a Signa mierocentrifuge (West Germany) model 202MK in 1.5 mls snap top microcentrifuge tubes for 5 minutes at 10,000 rpm. The cell pellet was resuspended very gently by pipeting in a room temperature sucrose solution (20% sucrose w/v, 30 mM Tris-Hcl pH7.5, 1 mM EDTA), using 1 μl/1 Klett unit. Following a 10 minute incubation at room temperature, the cells were centrifuged for 5 minutes at 10,000 rpm. The sucrose fraction was carefully removed from the cell pellet. The cell pellet was then resuspended very gently by pipeting in ice cold distilled water, using 1 μl/1 Klett unit. Following a 10 minute incubation on ice, the cells were centrifuged for 5 minutes at 12,000 rpm. The water fraction was carefully removed. Equal volumes of the sucrose and water fractions were pooled and aliquoted to provide samples for ELISA and biological activity screening.

Analysis of Protein Content of

E. coli

Cultures Producing hTL-3 Mutant Polypeptides

Bacterial cells from cultures treated as described above were collected from the medium by centrifugation. Aliquots of these cells were resuspended in SDS loading. buffer (4×: 6 g SDS, 10 ml beta-mercaptoethanol, 25 ml upper Tris gel stock (0.5 M Tris HCl pH 6.8, 0.4% SDS) brought to 50 ml with glycerol, 0.2% bromophenol blue was added) at a concentration of one microliter per Klett unit. These samples were incubated at 85° C. for five minutes and vortexed. Five or ten microliter aliquots of these samples were loaded on 15% polyacrylamide gels prepared according to the method of Laemmli (1970). Protein bands were visualized by staining the gels with a solution of acetic acid, methanol and water at 5:1:5 (volume to volume) ratio to which Coomassie blue had been added to a final concentration of 1%. After staining, the gels were washed in the same solution without the Coomassie blue and then washed with a solution of 7% acetic acid, 5% methanol. Gels were dried on a gel drier Model SE1160 obtained from Hoeffer (San Francisco, Calif.). The amount of stained protein was measured using a densitometer obtained from Joyce-Loebl (Gateshead, England). The values obtained were a measure of the amount of the stained hIL-3 protein compared to the total of the stained protein of the bacterial cells.

Western Blot Analysis of hTL-3 Muteins Made in

E. coli

In some

E. coli

cultures producing hIL-3, the level of accumulation of the hIL-3 protein is lower than 5% of total bacterial protein. To detect hIL-3 produced at this level, Western blot analysis was used. Proteins from cultures induced with nalidixic acid or arabinose. were run on polyacrylamide gels as described above except that volumes of sample loaded were adjusted to produce appropriate signals. After electrophoresis, the proteins were electroblotted to APT paper, Transa-bind, Schleicher and Schuell (Keene, N.H.) according to the method of Renart et al. (1979). Antisera used to probe these blots had been raised in rabbits, using peptides of the sequence of amino acids 20 to 41 and 94 to 118 of hIL-3 as the immunogens. The presence of bound antibody was detected with Staphylococcal protein A radiolabeled with

125

I, obtained from New England Nuclear (Boston, Mass.).

Fractionation of

E. coli

Cells Producing hIL-3 Proteins in the Cytoplasm

Cells from

E. coli

cultures harboring plasmids that produce hIL-3 muteins were induced with nalidixic acid. After three hours, the hIL-3 muteins accumulated in refractile bodies. The first step in purification of the hIL-3 muteins was to sonivate cells. Aliquots of the culture were resuspended from cell pellets in sonication buffer: 10 mM Tris, pH 8.0, 1 mM EDTA, 50 mM NaCl and 0.1 mM PMSF. These resuspended cells were subjected to several repeated sonication bursts using the microtip from a Sonicator cell disrupter, Model W-375 obtained from Heat Systems-Ultrasonics Inc. (Farmingdale, N.Y.). The extent of sonication was monitored by examining the homogenates under a light microscope. When nearly all of the cells had been broken, the homogenates were fractionated by centrifugation. The pellets, which contain most of the refractile bodies, are highly enriched for hIL-3 muteins.

Methods: Extraction, Refolding and Purification of Interleukin-3 (IL-3) Muteins Expressed as Refractile Bodies in

E. coli.

Extraction of refractile bodies (RB's):

For each gram of RB's (and typically one gram is obtained from a 300 ml

E. coli

culture), 5 ml of a solution containing 6M guanidine hydrochloride (GnHCl), 50 mM 2-N-cyclohexylaminoethanesulfonic acid (CHES) pH 9.5 and 20 mM dithiothreitol (DTT) was added. The RB's were extracted with a Bio-Homogenizer for 15-30 seconds and gently rocked for 2 hours at 5 degrees centigrade (5° C.) to allow the protein to completely reduce and denature.

Refolding of the IL-3 Muteins

The protein solution was transferred to dialysis tubing (1000 molecular weight cut-off) and dialyzed against at least 100 volumes of 4M GnHCl-50 mM CHES pH 8.0. The dialysis was continued overnight at 5° C. while gently stirring. Subsequently dialysis was continued against at least 100 volumes of 2M GnHCl-50 mM CHES pH 8.0 and dialyzed overnight at 5° C. while gently stirring.

Purification of the IL-3 Muteins

The protein solution was removed from the dialysis tubing and acidified by the addition of 40% acetonitrile (CH

3

CN)-0.2% trifluoroacetic acid (TFA) to a final concentration of 20% CH

3

CN-0.1% TFA. This was centrifuged (16,000×g for 5 minutes) to clarify and the supernatant was loaded onto a Vydac C-18 reversed phase column (10×250 mm) available from Vydac (Hesperia, Calif.) previously equilibrated in 20% CH

3

CN-0.1% TFA. The column was eluted with a linear gradient (0.2% CH

3

CN/minute) between 40-50% CH

3

CN-0.1% TFA at a flow rate of 3 ml/minute while collecting 1.5 ml fractions. The fractions were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and the appropriate fractions pooled. The pooled material was dried by lyophilization or in a Speed Vac concentrator. The dry powder was reconstituted with 10 mM ammonium bicarbonate pH 7.5, centrifuged (16,000×g for 5 minutes) to clarify and assayed for protein concentration by the method of Bradford (1976) with bovine serum albumin as the standard. Such protein can be further analyzed by additional techniques such as, SDS-PAGE, electrospray mass spectrometry, reverse phase HPLC, capillary zone electrophoresis, amino acid composition analysis, and ELISA (enzyme-linked immunosorbent assay).

hIL-3 Sandwich ELISA

IL-3 protein concentrations were determined using a sandwich ELISA based on an affinity purified polyclonal goat anti-rhIL-3. Microtiter plates (Dynatech Immulon II) were coated with 150 μl goat-anti-rhIL-3 at a concentration of approximately 1 μg/ml in 100 mM NaHCO3, pH 8.2. Plates were incubated overnight at room temperature in a chamber maintaining 100% humidity. Wells were emptied and the remaining reactive sites on the plate were blocked with 200 μl of solution containing 10 mM PBS, 3% BSA and 0.05% Tween 20, pH 7.4 for 1 hour at 37° C. and 100% humidity. Wells were emptied and washed 4× with 150 mM NaCl containing 0.05% Tween 20 (wash buffer). Each well then received 150 μl of dilution buffer (10 mM PBS containing 0.1% BSA, 0.01% Tween 20, pH 7.4), containing rhIL-3 standard, control, sample or dilution buffer alone. A standard curve was prepared with concentrations ranging from 0.125 ng/ml to 5 ng/ml using a stock solution of rhIL-3 (concentration determined by amino acid composition analysis). Plates were incubated 2,5 hours at 37° C. and 100% humidity. Wells were emptied and each plate was washed 4× with wash buffer. Each well then received 150 μl of an optimal dilution (as determined in a checkerboard assay format) of goat anti-rhIL-3 conjugated to horseradish peroxidase. Plates were incubated 1.5 hours at 37° C. and 100% humidity. Wells were emptied and each plate was washed 4× with wash buffer. Each well then received 150 ul of ABTS substrate solution (Kirkegaard and Perry). Plates were incubated at room temperature until the color of the standard wells containing 5 ng/ml rhIL-3 had developed enough to yield an absorbance between 0.5-1.0 when read at a test wavelength of 410 nm and a reference wavelength of 570 nm on a Dynatech microtiter plate reader. Concentrations of immunoreactive rhIL-3 in unknown samples were calculated from the standard curve using software supplied with the plate reader.

AML Prolifpraton Assay for Bioactive Human Interleukkin-3

The factor-dependent cell line AML 193 was obtained from the American Type Culture Collection (ATCC, Rockville, Md.). This cell line, established from a patient with acute myelogenous leukemia, is a growth factor dependent cell line which displayed enhanced growth in GM/CSF supplemented medium (Lange, B., et al., (1987); Valtieri, M., et al., (1987). The ability of AML 193 cells to proliferate in the presence of human IL-3 has also been documented. (Santoli, D., et al., (1987)). A cell line variant was used, AML 193 1.3, which was adapted for long term growth In IL-3 by washing out the growth factors and starving the cytokine dependent AML 193 cells for growth factors for 24 hours. The cells were then replated at 1×10

5

cells/well in a 24 well plate in media containing 100 U/ml IL-3. It took approximately 2 months for the cells to grow rapidly in IL-3. These cells were maintained as AML 193 1.3 thereafter by supplementing tissue culture medium (see below) with human IL-3.

AML 193 1.3 cells were washed 6 times in cold Hanks balanced salt solution (HBSS, Gibco, Grand Island, N.Y.) by centrifuging cell suspensions at 250×g for 10 minutes followed by decantation of supernatant. Pelleted cells were resuspended in HBSS and the procedure was repeated until six wash cycles were completed. Cells washed six times by this procedure were resuspended in tissue culture medium at a density ranging from 2×10

5

to 5×10

5

viable cells/ml. This medium was prepared by supplementing Iscove's modified Dulbeccols Medium (IMDM, Hazleton, Lenexa, Kans.) with albumin, transferrin, lipids and 2-mercaptoethanol. Bovine albumin (Boehringer-Mannheim, Indianapolis, Ind.) was added at 500 μg/ml; human transferrin (Boehringer-Mannheim, Indianapolis, Ind.) was added at 100 μg/ml; soybean lipid (Boehringer-Mannheim, Indianapolis, Ind.) was added at 50 μg/ml; and 2-mercaptoethanol (Sigma, St. Louis, Mo.) was added at 5×10

−5

M.

Serial dilutions of human interleukin-3 or human interleukin-3 variant protein (hIL-3 mutein) were made in triplicate series in tissue culture medium supplemented as stated above in 96 well Costar 3596 tissue culture plates. Each well contained 50 gl of medium containing interleukin-3 or interleukin-3 variant protein once serial dilutions were completed. Control wells contained tissue culture medium alone (negative control). AMM 193 1.3 cell suspensions prepared as above were added to each well by pipetting 50 μl (2.5×10

4

cells) into each well. Tissue culture plates were incubated at 37° C. with 5% CO

2

in humidified air for 3 days. On day 3, 0.5 μCi

3

H-thymidine (2 Ci/mM, New England Nuclear, Boston, Mass.) was added in 50 μl of tissue culture medium. Cultures were incubated at 37° C. with 5% CO

2

in humidified air for 18-24 hours. Cellular DNA was harvested onto glass filter mats (Pharmacia LKB, Gaithersburg, Md.) using a TOMTEC cell harvester (TOMTEC, Orange, Conn.) which utilized a water wash cycle followed by a 70% ethanol wash cycle. Filter mats were allowed to air dry and then placed into sample bags to which scintillation fluid (Scintiverse II, Fisher Scientific, St. Louis, Mo. or BetaPlate Scintillation Fluid, Pharmacia LKB, Gaithersburg, Md.) was added. Beta emissions of samples from individual tissue culture wells were counted in a LKB Betaplate model 1205 scintillation counter (Pharmacia LKB, Gaithersburg, Md.) and data was expressed as counts per minute of

3

H-thymidine incorporated into cells from each tissue culture well. Activity of each human interleukin-3 preparation or human interleukin-3 variant preparation was quantitated by measuring cell proliferation (

3

H-thymidine incorporation) induced by graded concentrations of interleukin-3 or interleukin-3 variant. Typically, concentration ranges from 0.05 pM-10

5

pM are quantitated in these assays. Activity is determined by measuring the dose of interleukin-3 or interleukin-3 variant which provides 50% of maximal proliferation [EC

50

=0.5× (maximum average counts per minute of

3

H-thymidine incorporated per well among triplicate cultures of all concentrations of interleukin-3 tested - background proliferation measured by

3

H-thymidine incorporation observed in triplicate cultures lacking interleukin-3]. This EC

50

value is also equivalent to 1 unit of bioactivity. Every assay was performed with native interleukin-3 as a reference standard so that relative activity levels could be assigned.

Relative biological activities of some IL-3 muteins of the present invention are shown in Table 1. The Relative Biological Activity of IL-3 mutants is calculated by dividing the EC

50

of (1-133) hIL-3 by the EC

50

of the mutant. The Relative Biological Activity may represent the average of replicate assays.

TABLE 1

BIOLOGICAL ACTIVITY OF IL-3 MUTEINS

Relative

Plasmid

Polypeptide

Biological

Code

Structure

Activity

Reference

(1-133)hIL-3

1.0

pMON13286

[SEO ID NO. 69]

8.0

pMON13304

[SEO ID NO. 66]

3.2

* The Relative Biological Activity of IL-3 mutants is calculated by dividing the EC

50

of (1-133) hIL-3 by the EC

50

of the mutant.

The following assay is used to measure IL-3 mediated sulfidoleukotriene release from human mononuclear cells.

IL-3 Mediated Sulfidoleukotriene Release from Human Mononuclear Cells

Heparin-containing human blood was collected and layered onto an equal volume of Ficoll-Paque (Pharmacia #17-0840-02) ready to use medium (density 1.077 g/ml.). The Ficoll was warmed to room temperature prior to use and clear 50 ml polystyrene tubes were utilized. The Ficoll gradient was spun at 300× g for 30 minutes at room temperature using a H1000B rotor in a Sorvall RT6000B refrigerated centrifuge. The band containing the mononuclear cells was carefully removed, the volume adjusted to 50 mls with Dulbecco's phosphate-buffered saline (Gibco Laboratories cat. #310-4040PK), spun at 400×g for 10 minutes at 40° C. and the supernatant was carefully removed. The cell pellet was washed twice with HA Buffer [20 mM Hepes (Sigma # H-3375), 125 mM NaCl (Fisher # S271-500), 5 mM KCl (sigma # P-9541), 0.5 mM glucose (Sigma # G-5000), 0.025% Human Serum Albumin (Calbiochem #126654) and spun at 300×g, 10 min., 4° C. The cells were resuspended in HACM Buffer (HA buffer supplemented with 1 mM CaC12 (Fisher # C79-500) and 1 mM MgC12 (Fisher # M-33) at a concentration of 1×106 cells/ml and 180 μl were transferred into each well of 96 well tissue culture plates. The cells were allowed to acclimate at 37° C. for 15 minutes. The cells were primed by adding 10 μls of a 20× stock of various concentrations of cytokine to each well (typically 100000, 20000, 4000, 800, 160, 32, 6.4, 1.28, 0 fM IL3). The cells were incubated for 15 minutes at 37° C. Sulfidoleukotriene release was activated by the addition of 10 μl of 20×(1000 nM) fmet-leu-phe (Calbiochem #344252) final concentration 50 nM FMLP and incubated for 10 minutes at 37° C. The plates were spun at 350×g at 4° C. for 20 minutes. The supernatants were removed and assayed for sulfidoleukotrienes using Cayman's Leukotriene C4 EIA kit (Cat. #420211) according to manufacturers' directions. Native (15-125) hIL-3 was run as a standard control in each assay.

Native hIL-3 possesses Considerable inflammatory activity and has been shown to stimulate synthesis of the arachidonic acid metabolites LTC

4

, LTD

4

, and LTE

4

; histamine synthesis and histamine release. Human clinical trials with native hIL-3 have documented inflammatory responses (Biesma, et al., BLOOD, 80:1141-1148 (1992) and Postmus, et al., J. CLIN. ONCOL., 0:1131-1140 (1992)). A recent study indicates that leukotrienes are involved in IL-3 actions in vivo and may contribute significantly to the biological effects of IL-3 treatment (Denzlinger, C., et al., BLOOD, 81:2466-2470 (1993))

Some muteins of the present invention may have an improved therapeutic profile as compared to native hIL-3 or (15-125) hIL-3. For example, some muteins of the present invention may have a similar or more potent growth factor activity relative to native hIL-3 or (15-125) hIL-3 without having a similar or corresponding increase in the stimulation of leukotriene or histamine. These muteins would be expected to have a more favorable therapeutic profile since the amount of polypeptide which needs to be given to achieve the desired growth factor activity (e. g. cell proliferation) would have a lesser leukotriene or histamine stimulating effect. In studies with native hIL-3, the stimulation of inflammatory factors has been an undesirable side effect of the treatment, Reduction or elimination of the stimulation of mediators of inflammation would provide an advantage over the use of native hIL-3.

Some muteins of the present invention may have antigenic profiles which differ from that of native hIL-3. For example, in a competition ELISA with an affinity purified polyclonal goat anti-hIL-3 antibody, native hIL-3 significantly blocked the binding of labeled hIL-3 to polyclonal anti-hIL-3 antibody. Some polypeptides of the present invention, particularly those with several amino acids differing from those of native hIL-3, fail to block the binding of hIL-3 to anti-hIL-3 antibody.

Table 2 lists the sequences of some oligonucleotides used in making the muteins of the present invention.

Table 3 lists the amino acid sequence of native (15-125) hIL-3 (Peptide #1) and the amino acid sequences of some mutant polypeptides of the present invention. The sequences are shown with the amino acid numbering corresponding to that of native hIL-3 [FIG.

1

].

TABLE 2

OLIGONUCLEOTIDES

Oligo #1

AATTCCGTCG TAAACTGACC TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAGT

[SEQ ID NO: 8]

AATA

Oligo #2

AGCTTATTAC TGTTGAGCCT GCGCGTTCTC CAAGGTTTTC AGATAGAAGG TCAGTTTACG

[SEQ ID NO: 9]

ACGG

Oligo #3

CTAGCCACGG CCGCACCCAC GCGACATCCA ATCCATATCA AGGACGGTGA CTGGAATG

[SEQ ID NO:24]

Oligo #4

TTAACATTCC AGTCACCGTC CTTGATATGG ATTGGATGTC GCGTGGGTGC GGCCGTGG

[SEQ ID NO:25]

Oligo #5

CATGGCTAAC TGCTCTAACA TGAT

[SEQ ID NO:151]

Oligo #6

CGATCAT GTTAGAGCAGTTAGC

[SEQ ID NO:152]

Oligo #7 IL3MUTNCO

TGTCTGCTCA GGCCATGGCT

[SEQ ID NO:26]

Oligo #8 IL3T93

GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGTCGAC TTATTACGTG GGTGCGGCCG

[SEQ ID NO:27]

TGGCTAG

Oligo #9 IL3T97

GCGCGAATTC ATTCCAGTCA CCGTCGACTT ATTAGATTGG ATGTCGCGTG GGTGC

[SEQ ID NO:28]

Oligo #10 IL3T101

GCGCGAATTC GTCGACTTAT TAGTCCTTGA TATGGATTGG ATG

[SEQ ID NO:31]

Oligo #11 IL3R94

GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATTGG ATG

SNN

CGTG GGTGCGGCCG

[SEQ ID NO:32]

TGGCTAG

Oligo #12 IL3R95

GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATTGG

SNN

TCGCGTG GGTGCGGCCG

[SEQ ID NO:33]

TGGC

Oligo #13 IL3R96

GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGAT

SNN

ATGTCGCGTG GGTGCGGCCG

[SEQ ID NO:34]

T

Oligo #14 IL3R97

GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATG

SNN

TGG ATGTCGCGTG GGTGCGGC

[SEQ ID NO:35]

Oligo #15 IL3P9497

GATATGGATT GGATGTCGCG TGGG

[SEQ ID NO:36]

Oligo #16 IL3R98

GCGCGAATTC ATTCCAGTCA CCGTCCTTGA T

SNN

GATTGG ATGTCGCGTG GGTGC

[SEQ ID NO:37]

Oligo #17 IL3R99

GCGCGAATTC ATTCCAGTCA CCGTCCTT

SN N

ATGGATTGG ATGTCGCGTG GG

[SEQ ID NO:38]

Oligo #18 IL3R100

GCGCGAATTC ATTCCAGTCA CCGTC

SNN

GA TATGGATTGG ATGTCGCGT

[SEQ ID NO:39]

Oligo #19 IL3R101

GCGCGAATTC ATTCCAGTCA CC

SNN

CTTGA TATGGATTGG ATGTCG

[SEQ ID NO:40]

Oligo #20 IL3P98100

GTCACCGTCC TTGATATGGA TTGG

[SEQ ID NO:41]

Oligo #21 IL3R102

GCGCGAATTC ATTCCAGTC

S NN

GTCCTTGA TATGGATTGG ATG

[SEQ ID NO:42]

Oligo #22 IL3R103

GCGCGAATTC ATTCCA

SNN

A CCGTCCTTGA TATGGATTGG

[SEQ ID NO:43]

Oligo #23 IL3R104

GCGCGAATTC ATT

SNN

GTCA CCGTCCTTGA TATGGAT

[SEQ ID NO:44]

Oligo #24 IL3R105

GCGCGAATTC

SNN

CCAGTCA CCGTCCTTGA TATG

[SEQ ID NO:45]

Oligo #25 IL3P102105

GAATTCATTC CAGTCACCGT TCCTT

[SEQ ID NO:46]

Oligo #26 IL3MUTR1

CGCGCGGAAT TCATTCCAGT CACCGT

[SEQ ID NO:47]

Oligo #27 DEL1722

CGCGCGCCAT GGCTAACTGC ATTATAACAC ACACTTAAAG CA

[SEQ ID NO:48]

Oligo #28 DEL2328

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAACA GCCACCTTTG CCTTTGCT

[SEQ ID NO:49]

Oligo #29 DEL2934

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCTGG

[SEQ ID NO:50]

ACTTCAACAA CCTCAA

Oligo #30 DEL3540

GCGCGCGATA TCTTGGTCTT CTTCACCATT CAGCGGCAGC GGTGGCTGCT

[SEQ ID NO:51]

Oligo #31 DEL4146

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATGAGGT TGTTGAAGTC

[SEQ ID NO:52]

CAGCA

Oligo #32 DEL4752

GCGCGCCTCG AGGTTTGGAC GACGAAGATC TTGGTCTTCA CCATTGA

[SEQ ID NO:53]

Oligo #33 DEL5358

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGT TATTTTCCAT

[SEQ ID NO:54]

CAGGATAT

Oligo #34 DEL5964

GCGCGCTGAT GCATTCTGCA GAGACTTGAC GAGGTTTGGA CGACGAAGGT

[SEQ ID NO:55]

Oligo #35 DEL6570

GCGCGCCTCG AGGCATTCAA CCGTGCTGCA TCAGCAATTG AGAGCAT

[SEQ ID NO:56]

Oligo #36 DEL7176

GCGCGCCTGC AGAATATTCT TAAAAATCTC CTGCC

[SEQ ID NO:57]

Oligo #37 DEL7782

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCCCATGTC TGCCGCTAGC CAC

[SEQ ID NO:58]

Oligo #38 DEL8388

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GACGGCCGCA

[SEQ ID NO:59]

CCCACGCGAC A

Oligo #39 DEL8893

CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCA GGGCAGACAT

[SEQ ID NO:60]

GGCAGGA

Oligo #40 DEL106111

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTAT

[SEQ ID NO:61]

TCCAGTCACC GTCCTTGA

Oligo #41 DEL112117

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA CAGTTTACGA CGGAATTCAT

[SEQ ID NO:62]

Oligo #42 DEL118123

CGCGCGAAGC TTATTACTGT TGGGTTTTCA GATAGAAGGT CA

[SEQ ID NO:63]

Oligo #43 R17IL3 Length: 000058

CGCGCGCCAT GGCTAACTGC NNSAACATGA TCGATGAAAT TATAACACAC TTAAAGCA

[SEQ ID NO:64]

Oligo #44 R18IL3 Length: 000058

CGCGCGCCAT GGCTAACTGC TCTNNSATGA TCGATGAAAT TATAACACAC TTAAAGCA

[SEQ ID NO:222]

Oligo #45 R19IL3 Length: 000058

CGCGCGCCAT GGCTAACTGC TCTAACNNSA TCGATGAAAT TATAACACAC TTAAAGCA

[SEQ ID NO:223]

Oligo #46 R20IL3 Length: 000058

CGCGCGCCAT GGCTAACTGC TCTAACATGN NSGATGAAAT TATAACACAC TTAAAGCA

[SEQ ID NO:224]

Oligo #47 R21IL3 Length: 000058

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCNNSGAAAT TATAACACAC TTAAAGCA

[SEQ ID NO:225]

Oligo #48 R22IL3 Length: 000058

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATNNSAT TATAACACAC TTAAAGCA

[SEQ ID NO:226]

Oligo #49 R23IL3 Length: 000076

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAANN SATAACACAC TTAAAGCAGC

[SEQ ID NO:227]

CACCTTTGCC TTTGCT

Oligo #50 R24IL3 Length: 000076

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TNNSACACAC TTAAAGCAGC

[SEQ ID NO:228]

CACCTTTGCC TTTGCT

Oligo #51 R25IL3 Length: 000076

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATANNSCAC TTAAAGCAGC

[SEQ ID NO:229]

CACCTTTGCC TTTGCT

Oligo #52 R26IL3 Length: 000076

CGCGCGCCAT GGCTAACTCC TCTAACATGA TCGATGAAAT TATAACANNS TTAAAGCAGC

[SEQ ID NO:74]

CACCTTTGCC TTTGCT

Oligo #53 R27IL3 Length: 000076

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC NNSAAGCAGC

[SEQ ID NO:75]

CACCTTTGCC TTTGCT

Oligo #54 R28IL3 Length: 000076

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTANNSCAGC

[SEQ ID NO:76]

CACCTTTGCC TTTGCT

Oligo #55 R29IL3 Length: 000094

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGNNSC

[SEQ ID NO:77]

CACCTTTGCC TTTGCTGGAC TTCAACAACC TCAA

Oligo #56 R30IL3 Length: 000094

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGN

[SEQ ID NO:78]

NSCCTTTGCC TTTGCTGGAC TTCAACAACC TCAA

Oligo #57 R31IL3 Length: 000094

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC

[SEQ ID NO:79]

CANNSTTGCC TTTGCTGGAC TTCAACAACC TCAA

Oligo #58 R32IL3 Length: 000094

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC

[SEQ ID NO:80]

CACCTNNSCC TTTGCTGGAC TTCAACAACC TCAA

Oligo #59 R33IL3 Length: 000094

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC

[SEQ ID NO:81]

CACCTTTGNN STTGCTGGAC TTCAACAACC TCAA

Oligo #60 R34IL3 Length: 000094

CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC

[SEQ ID NO:82]

CACCTTTGCC TNNSCTGGAC TTCAACAACC TCAA

Oligo #61 R35IL3 Length: 000065

GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTGAAG TCSNNCAGCG GCAGCGGTGG

[SEQ ID NO:83]

CTGCT

Oligo #62 R36IL3 Length: 000065

GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTGAAS NNCAGCAGCG GCAGCG

[SEQ ID NO:84]

GTGGCTGCT

Oligo #63 R37IL3 Length: 000065

GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTSNNG TCCAGCAGCG GCAGCGGTGG

[SEQ ID NO:85]

CTGCT

Oligo #64 R38IL3 Length: 000065

GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTSNNGAAG TCCAGCAGCG GCAGCGGTGG

[SEQ ID NO:86]

CTGCT

Oligo #65 R39IL3 Length: 000065

GCGCGCGATA TCTTGGTCTT CACCATTGAG SNNGTTGAAG TCCAGCAGCG GCAGCGGTGG

[SEQ ID NO:87]

CTGCT

Oligo #66 R40IL3 Length: 000065

GCGCGCGATA TCTTGGTCTT CACCATTSNN GTTGTTGAAG TCCAGCAGCG GCAGCGGTGG

[SEQ ID NO:88]

CTGCT

Oligo #67 R41IL3 Length: 000083

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCTTCACC

[SEQ ID NO:89]

SNNGAGGTTG TTGAAGTCCA GCA

Oligo #68 R42IL3 Length: 000083

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCTTCSNN

[SEQ ID NO:90]

ATTGAGGTTG TTGAAGTCCA GCA

Oligo #69 R43IL3 Length: 000083

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCSNNACC

[SEQ ID NO:91]

ATTGAGGTTG TTGAAGTCCA GCA

Oligo #70 R44IL3 Length: 000083

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GSNNTTCACC

[SEQ ID NO:92]

ATTGAGGTTG TTGAAGTCCA GCA

Oligo #71 R45IL3 Length: 000083

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCSN NGTCTTCACC

[SEQ ID NO:93]

ATTGAGGTTG TTGAAGTCCA GCA

Oligo #72 R46IL3 Length: 000083

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATSNNTT GGTCTTCACC

[SEQ ID NO:94]

ATTGAGGTTG TTGAAGTCCA GCA

Oligo #73 R47IL3 Length: 000065

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGSNNATCTT GGTCTTCACC

[SEQ ID NO:95]

ATTGA

Oligo #74 R48IL3 Length: 000065

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATS NNGATATCTT GGTCTTCACC

[SEQ ID NO:96]

ATTGA

Oligo #75 R49IL3 Length: 000065

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCSNNC AGGATATCTT GGTCTTCACC

[SEQ ID NO:97]

ATTGA

Oligo #76 R50IL3 Length: 000065

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTSNNCATC AGGATATCTT GGTCTTCACC

[SEQ ID NO:98]

ATTGA

Oligo #77 R51IL3 Length: 000065

GCGCGCCTCG AGGTTTGGAC GACGAAGGTT SNNTTCCATC AGGATATCTT GGTCTTCACC

[SEQ ID NO:99]

ATTGA

Oligo #78 R52IL3 Length: 000065

GCGCGCCTCG AGGTTTGGAC GACGAAGSNN ATTTTCCATC AGGATATCTT GGTCTTCACC

[SEQ ID NO:100]

ATTGA

Oligo #79 R53IL3 Length: 000086

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGACG

[SEQ ID NO:101]

ACGSNNGTTA TTTTCCATCA GGATAT

Oligo #80 R54IL3 Length: 000086

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGACG

[SEQ ID NO:102]

SNNAAGGTTA TTTTCCATCA GGATAT

Oligo #81 R55IL3 Length: 000086

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGSNN

[SEQ ID NO:103]

ACGAAGGTTA TTTTCCATCA GGATAT

Oligo #82 R56IL3 Length: 000086

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTSNNACG

[SEQ ID NO:104]

ACGAAGGTTA TTTTCCATCA GGATAT

Oligo #83 R57IL3 Length: 000086

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GSNNTGGACG

[SEQ ID NO:105]

ACGAAGGTTA TTTTCCATCA GGATAT

Oligo #84 R58IL3 Length: 000086

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCSN NGTTTGGACG

[SEQ ID NO:106]

ACGAAGGTTA TTTTCCATCA GGATAT

Oligo #85 R59IL3 Length: 000068

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCSNNGA GGTTTGGACG

[SEQ ID NO:107]

ACGAAGGT

Oligo #86 R60IL3 Length: 000068

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AASNNCTCGA GGTTTGGACG

[SEQ ID NO:108]

ACGAAGGT

Oligo #87 R61IL3 Length: 000068

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTS NNTGCCTCGA GGTTTGGACG

[SEQ ID NO:109]

ACGAAGGT

Oligo #88 R62IL3 Length: 000068

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGSNNG AATGCCTCGA GGTTTGGACG

[SEQ ID NO:110]

ACGAAGGT

Oligo #89 R63IL3 Length: 000068

GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCSNNGTTG AATGCCTCGA GGTTTGGACG

[SEQ ID NO:111]

ACGAAGGT

Oligo #90 R64IL3 Length: 000068

GCGCGCTGAT GCATTCTGCA GAGACTTGAC SNNACGGTTG AATGCCTCGA GGTTTGGACG

[SEQ ID NO:112]

ACGAAGGT

Oligo #91 R65IL3 Length: 000065

GCGCGCCTCG AGGCATTCAA CCGTGCTNNS AAGTCTCTGC AGAATGCATC AGCAATTGAG

[SEQ ID NO:113]

AGCAT

Oligo #92 R66IL3 Length: 000065

GCGCGCCTCG AGGCATTCAA CCGTGCTGTC NNSTCTCTGC AGAATGCATC AGCAATTGAG

[SEQ ID NO:114]

AGCAT

Oligo #93 R67IL3 Length: 000065

GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGNNSCTGC AGAATGCATC AGCAATTGAG

[SEQ ID NO:115]

AGCAT

Oligo #94 R68IL3 Length: 000065

GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTNNSC AGAATGCATC AGCAATTGAG

[SEQ ID NO:116]

AGCAT

Oligo #95 R69IL3 Length: 000065

GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTCTGN NSAATGCATC AGCAATTGAG

[SEQ ID NO:117]

AGCAT

Oligo #96 R70IL3 Length: 000065

GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTCTGC AGNNSGCATC AGCAATTGAG

[SEQ ID NO:118]

AGCAT

Oligo #97 R71IL3 Length: 000053

GCGCGCCTGC AGAATNNSTC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCC

[SEQ ID NO:119]

Oligo #98 R72IL3 Length: 000053

GCGCGCCTGC AGAATGCANN SGCAATTGAG AGCATTCTTA AAAATCTCCT GCC

[SEQ ID NO:120]

Oligo #99 R73IL3 Length: 000053

GCGCGCCTGC AGAATGCATC ANNSATTGAG AGCATTCTTA AAAATCTCCT GCC

[SEQ ID NO:121]

Oligo #100 R74IL3 Length: 000053

GCGCGCCTGC AGAATGCATC AGCANNSGAG AGCATTCTTA AAAATCTCCT GCC

[SEQ ID NO:122]

Oligo #101 R75IL3 Length: 000053

GCGCGCCTGC AGAATGCATC AGCAATTNNS AGCATTCTTA AAAATCTCCT GCC

[SEQ ID NO:123]

Oligo #102 R76IL3 Length: 000053

GCGCGCCTGC AGAATGCATC AGCAATTGAG NNSATTCTTA AAAATCTCCT GCC

[SEQ ID NO:124]

Oligo #103 R77IL3 Length: 000071

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCNNSCTTA AAAATCTCCT GCCATGTCTG

[SEQ ID NO:125]

CCGCTAGCCA C

Oligo #104 R78IL3 Length: 000071

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTNNSA AAAATCTCCT GCCATGTCTG

[SEQ ID NO:126]

CCGCTAGCCA C

Oligo #105 R79IL3 Length: 000071

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTN NSAATCTCCT GCCATGTCTG

[SEQ ID NO:127]

CCGCTAGCCA C

Oligo #106 R80IL3 Length: 000071

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AANNSCTCCT GCCATGTCTG

[SEQ ID NO:138]

CCGCTAGCCA C

Oligo #107 R81IL3 Length: 000071

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATNNSCT GCCATGTCTG

[SEQ ID NO:139]

CCGCTAGCCA C

Oligo #108 R82IL3 Length: 000071

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCNN SCCATGTCTG

[SEQ ID NO:140]

CCGCTAGCCA C

Oligo #109 R83IL3 Length: 000089

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GNNSTGTCTG

[SEQ ID NO:141]

CCGCTAGCCA CGGCCGCACC CACGCGACA

Oligo #110 R84IL3 Length: 000089

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCANNSCTG

[SEQ ID NO:142]

CCGCTAGCCA CGGCCGCACC CACGCGACA

Oligo #111 R85IL3 Length: 000089

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTNNS

[SEQ ID NO:143]

CCGCTAGCCA CGGCCGCACC CACGCGACA

Oligo #112 R86IL3 Length: 000089

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG

[SEQ ID NO:157]

NNSCTAGCCA CGGCCGCACC CACGCGACA

Oligo #113 R87IL3 Length: 000089

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG

[SEQ ID NO:158]

CCGNNSGCCA CGGCCGCACC CACGCGACA

Oligo #114 R88IL3 Length: 000089

GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG

[SEQ ID NO:159]

CCGCTANNSA CGGCCGCACC CACGCGACA

Oligo #115 R89IL3 Length: 000086

CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGTGCGGC

[SEQ ID NO:160]

SNNGGCCAGG GCCAGACATG GCAGGA

Oligo #116 R90IL3 Length: 000086

CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGTGCSNN

[SEQ ID NO:161]

CGTGGCCAGG GGCAGACATG GCAGGA

Oligo #117 R91IL3 Length: 000086

CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGSNNGGC

[SEQ ID NO:162]

CGTGGCCAGG GGCAGACATG GCAGGA

Oligo #118 R92IL3 Length: 000086

CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TSNNTGCGGC

[SEQ ID NO:163]

CGTGGCCAGG GGCAGACATG GCAGGA

Oligo #119 R93IL3 Length: 000086

CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGSN NGGGTGCGGC

[SEQ ID NO:164]

CGTGGCCAGG GGCAGACATG GCAGGA

Oligo #120 3PR106 Length: 000048

TTTCAGATAG AAGGTCAGTT TACGACGGAA SNNATTCCAG TCACCGTC

[SEQ ID NO:165]

Oligo #121 3PR107 Length: 000048

TTTCAGATAG AAGGTCAGTT TACGACGSNN TTCATTCCAG TCACCGTC

[SEQ ID NO:166]

Oligo #122 3PR108 Length: 000048

TTTCAGATAG AAGGTCAGTT TACGSNNGAA TTCATTCCAG TCACCGTC

[SEQ ID NO:167]

Oligo #123 3PR109 Length: 000048

TTTCAGATAG AAGGTCAGTT TSNNACGGAA TTCATTCCAG TCACCGTC

[SEQ ID NO:168]

Oligo #124 3PR110 Length: 000048

TTTCAGATAG AAGGTCAGSN NACGACGGAA TTCATTCCAG TCACCGTC

[SEQ ID NO:169]

Oligo #125 3PR111 Length: 000048

TTTCAGATAG AAGGTSNNTT TACGACGGAA TTCATTCCAG TCACCGTC

[SEQ ID NO:170]

Oligo #126 IL3MUTD3 Length: 000023

CGCGCGAAGC TTATTACTGT TGA

[SEQ ID NO:171]

Oligo #127 R112IL3 Length: 000078

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TAGAASNNCA

[SEQ ID NO:172]

GTTTACGACG GAATTCAT

Oligo #128 R113IL3 Length: 000078

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TASNNGGTCA

[SEQ ID NO:173]

GTTTACGACG GAATTCAT

Oligo #129 R114IL3 Length: 000078

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGS NNGAAGGTCA

[SEQ ID NO:174]

GTTTACGACG GAATTCAT

Oligo #130 R115IL3 Length: 000078

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTSNNA TAGAAGGTCA

[SEQ ID NO:175]

GTTTACGACG GAATTCAT

Oligo #131 R116IL3 Length: 000078

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTSNNCAGA TAGAAGGTCA

[SEQ ID NO:176]

GTTTACGACG GAATTCAT

Oligo #132 R117IL3 Length: 000078

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA SNNTTTCAGA TAGAAGGTCA

[SEQ ID NO:177]

GTTTACGACG GAATTCAT

Oligo #133 R118IL3 Length: 000060

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCSNN GGTTTTCAGA TAGAAGGTCA

[SEQ ID NO:178]

Oligo #134 R119IL3 Length: 000060

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTSNNCAA GGTTTTCAGA TAGAAGGTCA

[SEQ ID NO:179]

Oligo #135 R120IL3 Length: 000060

CGCGCGAAGC TTATTACTGT TGAGCCTGCG CSNNCTCCAA GGTTTTCAGA TAGAAGGTCA

[SEQ ID NO:180]

Oligo #136 R121IL3 Length: 000060

CGCGCGAAGC TTATTACTGT TGAGCCTGSN NGTTCTCCAA GGTTTTCAGA TAGAAGGTCA

[SEQ ID NO:181]

Oligo #137 R122IL3 Length: 000060

CGCGCGAAGC TTATTACTGT TGAGCSNNCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTCA

[SEQ ID NO:182]

Oligo #138 R123IL3 Length: 000060

CGCGCGAAGC TTATTACTGT TGSNNCTGCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTCA

[SEQ ID NO:183]

Oligo #139 P1722IL3 Length: 000024

TGCTCTAACA TGATCGATGA AATT

[SEQ ID NO:184]

Oligo #140 P2328IL3 Length: 000024

GAAATTATAA CACACTTAAA GCAG

[SEQ ID NO:185]

Oligo #141 P2934IL3 Length: 000024

AAGCAGCCAC CTTTGCCTTT GCTG

[SEQ ID NO:186]

Oligo #142 P3540IL3 Length: 000024

AAGCAGCCAC CGCTGCCGCT GCTG

[SEQ ID NO:187]

Oligo #143 PRB41-46 Length: 000024

CTCAATGGTG AAGACCAAGA TATC

[SEQ ID NO:188]

Oligo #144 PRB47-52 Length: 000024

GATATCCTGA TGGAAAATAA CCTT

[SEQ ID NO:189]

Oligo #145 PRB53-58 Length: 000024

AACCTTCGTC GTCCAAACCT CGAG

[SEQ ID NO:190]

Oligo #146 PRB59-64 Length: 000024

CTCGAGGCAT TCAACCGTGC TGTC

[SEQ ID NO:191]

Oligo #147 PRB65-70 Length: 000024

GCTGTCAAGT CTCTGCAGAA TGCA

[SEQ ID NO:192]

Oligo #148 P7176IL3 Length: 000024

AATGCATCAG CAATTGAGAG CATT

[SEQ ID NO:193]

Oligo #149 P7782IL3 Length: 000024

AGCATTCTTA AAAATCTCCT GCCA

[SEQ ID NO:194]

Oligo #150 P8388IL3 Length: 000024

CTGCCATGTC TGCCCCTGGC CACG

[SEQ ID NO:195]

Oligo #151 P8893IL3 Length: 000024

CTGGCCACGG CCGCACCCAC GCGA

[SEQ ID NO:196]

Oligo #152 P106111 Length: 000024

AATGAATTCC GTCGTAAACT GACC

[SEQ ID NO:197]

Oligo #153 P112117 Length: 000024

CTGACCTTCT ATCTGAAAAC CTTG

[SEQ ID NO:198]

Oligo #154 P118123 Length: 000024

ACCTTGGAGA ACGCGCAGGC TCAA

[SEQ ID NO:199]

Oligo #155 PSTECRI1.REQ Length: 000022

GAATGCATCA GCAATTGAGA GC

[SEQ ID NO:200]

Oligo #156 PSTECRI5.REQ Length: 000020

AATTGCTGAT GCATTCTGCA

[SEQ ID NO:201]

Oligo #157 PSTECRI2.REQ Length: 000024

ATTCTTAAAA ATCTCCTGCC ATGT

[SEQ ID NO:202]

Oligo #158 PSTECRI6.REQ Length: 000024

CAGGAGATTT TTAAGAATGC TCTC

[SEQ ID NO:203]

Oligo #159 PSTECRI3.REQ Length: 000030

CTGCCCCTGG CCACGGCCGC ACCCACGCGA

[SEQ ID NO:204]

Oligo #160 PSTECRI7.REQ Length: 000030

GGGTGCGGCC GTGGCCAGGG GCAGACATGG

[SEQ ID NO:205]

Oligo #161 98I100R4.REQ Length: 000034

CATCCAATCA TCATCCGTGA CGGTGACTGG AATG

[SEQ ID No:206]

Oligo #162 98I100R8.REQ Length: 000044

AATTCATTCC AGTCACCGTC ACGGATGATG ATTGGATGTC GCGT

[SEQ ID NO:207]

Oligo 163 95R8I0R4.REQ Length: 000034

CGCCCAATCA TCATCCGTGA CGGTGACTGG AATG

[SEQ ID NO:208]

Oligo #164 95R8I0R8.REQ Length: 000044

AATTCATTCC AGTCACCGTC ACGGATGATG ATTGGGCGTC GCGT

[SEQ ID NO:209]

Oligo #165 NCOECRV1.REQ Length: 000040

CATGGCTAAC TGCTCTAACA TGATCGATGA AATTATAACA

[SEQ ID NO:210]

Oligo #166 NCOECRV4.REQ Length: 000045

CTTTAAGTGT GTTATAATTT CATCGATCAT GTTAGAGCAG TTAGC

[SEQ ID NO:211]

Oligo #167 NCOECRV2.REQ Length: 000036

CACTTAAAGC AGCCACCTTT GCCTTTGCTG GACTTC

[SEQ ID NO:212]

Oligo #168 NCOECRV5.REQ Length: 000036

GAGGTTGTTG AAGTCCAGCA AAGGCAAAGG TGGCTG

[SEQ ID NO:213]

Oligo #169 2D5M6SUP.REQ Length: 000027

AACAACCTCA ATGACGAAGA CATGTCT

[SEQ ID NO:214]

Oligo #170 2D5M6SLO.REQ Length: 000018

AGACATGTCT TCGTCATT

[SEQ ID NO:215]

Oligo #15(A) Length: 000016

TGAACCATAT GTCAGG

[SEQ ID NO:29]

Oligo #16(A) Length: 000024

AATTCCTGAC ATATGGTTCA TGCA

[SEQ ID NO:30]

Oligo #51(A) Length: 000034

GCCGATACCGCGGCATACTCCCACCATTCAGAGA

[SEQ ID NO:155]

Oligo #52(A) Length: 000033

GCCGATAAGATCTAAAACGGGTATGGAGAAACA

[SEQ ID NO:156]

Oligo #171 Length: 000040

CATGGCTAAC TGCTCTAACA TGATCAACGA AATTATAACA

[SEQ. ID NO:69]

Oligo #172 Length: 000045

CTTTAAGTGT GTTATAATTT CGTTGATCAT GTTAGAGCAG TTAGC

[SEQ. ID NO:70]

Oligo #173 Length: 000040

CATGGCTAAC TGCTCTAACA TGATCCAAGA AATTATAACA

[SEQ. ID NO:71]

Oligo #174 Length: 000045

CTTTAAGTGT GTTATAATTT CTTGGATCAT GTTAGAGCAG TTAGC

[SEQ. ID NO:72]

Oligo #175 Length: 000040

CATGGCTAAC TGCTCTAACA TGATCGAAGA AATTATAACA

[SEQ. ID NO:73]

Oligo #176 Length: 000045

CTTTAAGTGT GTTATAATTT CTTCGATCAT GTTAGAGCAG TTAGC

[SEQ. ID NO:219]

Oligo #177 Length: 000040

CATGGCTAAC TGCTCTAACA TGATCAGCGA AATTATAACA

[SEQ. ID NO:230]

Oligo #178 Length: 000045

CTTTAAGTGT GTTATAATTT CGCTGATCAT GTTAGAGCAG TTAGC

[SEQ. ID NO:231]

Oligo #179 Length: 000040

CATGGCTAAC TGCTCTAACA TGATCACCGA AATTATAACA

[SEQ. ID NO:232]

Oligo #180 Length: 000045

CTTTAAGTGT GTTATAATTT CCGTGATCAT GTTAGAGCAG TTAGC

[SEQ. ID NO:233]

Oligo #181 Length: 000040

CATGGCTAAC TGCTCTAACA TGATCGATAA CATTATAACA

[SEQ. ID NO:234]

Oligo #182 Length: 000045

CTTTAAGTGT GTTATAATGT TATCGATCAT GTTAGAGCAG TTAGC

[SEQ. ID NO:235]

Oligo #183 Length: 000040

CATGGCTAAC TGCTCTAACA TGATCGATGA CATTATAACA

[SEQ. ID NO:236]

Oligo #184 Length: 000045

CTTTAAGTGT GTTATAATGT CATCGATCAT GTTAGAGCAG TTAGC

[SEQ. ID NO:237]

Oligo #185 Length: 000040

CATGGCTAAC TGCTCTAACA TGATCGATCA GATTATAACA

[SEQ. ID NO:238]

Oligo #186 Length: 000045

CTTTAAGTGT GTTATAATCT GATCGATCAT GTTAGAGCAG TTAGC

[SEQ. ID NO:239]

Oligo #187 Length: 000040

CATGGCTAAC TGCTCTAACA TGATCGATCT GATTATAACA

[SEQ. ID NO:240]

Oligo #188 Length: 000045

CTTTAAGTGT GTTATAATCA GATCGATCAT GTTAGAGCAG TTAGC

[SEQ. ID NO:241]

Oligo #189 Length: 000040

CATGGCTAAC TGCTCTAACA TGATCGATGT TATTATAACA

[SEQ. ID NO:242]

Oligo #190 Length: 000045

CTTTAAGTGT GTTATAATAA CATCGATCAT GTTAGAGCAG TTAGC

[SEQ. ID NO:243]

Oligo #191 Length: 000036

CACTTAAAGC AGCCACCTTT GCCTGCTCTG GACTTC

[SEQ. ID NO:244]

Oligo #192 Length: 000036

GAGGTTGTTG AAGTCCAGAG CAGGCAAAGG TGGCTG

[SEQ. ID NO:245]

Oligo #193 Length: 000036

CACTTAAAGC AGCCACCTTT GCCTCGTCTG GACTTC

[SEQ. ID NO:246]

Oligo #194 Length: 000036

GAGGTTGTTG AAGTCCAGAC GAGGCAAAGG TGGCTG

[SEQ. ID NO:247]

Oligo #195 Length: 000036

CACTTAAAGC AGCCACCTTT GCCTCAGCTG GACTTC

[SEQ. ID NO:248]

Oligo #196 Length: 000036

GAGGTTGTTG AAGTCCAGCT GAGGCAAAGG TGGCTG

[SEQ. ID NO:249]

Oligo #197 Length: 000036

CACTTAAAGC AGCCACCTTT GCCTGAACTG GACTTC

[SEQ. ID NO:250]

Oligo #198 Length: 000036

GAGGTTGTTG AAGTCCAGCT CAGGCAAAGG TGGCTG

[SEQ. ID NO:251]

Oligo #199 Length: 000036

CACTTAAAGC AGCCACCTTT GCCTATCCTG GACTTC

[SEQ. ID NO:252]

Oligo #200 Length: 000036

GAGGTTGTTG AAGTCCAGGA TAGGCAAAGG TGGCTG

[SEQ. ID NO:253]

Oligo #201 Length: 000036

CACTTAAAGC AGCCACCTTT CCCTTTCCTG GACTTC

[SEQ. ID NO:254]

Oligo #202 Length: 000036

GAGGTTGTTG AAGTCCAGGA AAGGCAAAGG TGGCTG

[SEQ. ID NO:255]

Oligo #203 Length: 000036

CACTTAAAGC AGCCACCTTT GCCTACCCTG GACTTC

[SEQ. ID NO:256]

Oligo #204 Length: 000036

GAGGTTGTTG AAGTCCAGGG TAGGCAAAGG TGGCTG

[SEQ. ID NO:257]

Oligo #205 Length: 000027

AACAACCTCA ATCGTGAAGA CCAAGAT

[SEQ. ID NO:258]

Oligo #206 Length: 000018

ATCTTGGTCT TCACGATT

[SEQ. ID NO:259]

Oligo #207 Length: 000027

AACAACCTCA ATAACGAAGA CCAAGAT

[SEQ. ID NO:260]

Oligo #208 Length: 000018

ATCTTGGTCT TCGTTATT

[SEQ. ID NO:261]

Oligo #209 Length: 000027

AACAACCTCA ATGAAGAAGA CCAAGAT

[SEQ. ID NO:262]

Oligo #210 Length: 000018

ATCTTGGTCT TCTTCATT

[SEQ. ID NO:263]

Oligo #211 Length: 000027

AACAACCTCA ATATCGAAGA CCAAGAT

[SEQ. ID NO:264]

Oligo #212 Length: 000018

ATCTTGGTCT TCGATATT

[SEQ. ID NO:265]

Oligo #213 Length: 000027

AACAACCTCA ATCTGGAAGA CCAAGAT

[SEQ. ID NO:266]

Oligo #214 Length: 000018

ATCTTGGTCT TCCAGATT

[SEQ. ID NO:267]

Oligo #215 Length: 000027

AACAACCTCA ATAAAGAAGA CCAAGAT

[SEQ. ID NO:268]

Oligo #216 Length: 000018

ATCTTGGTCT TCTTTATT

[SEQ. ID NO:269]

Oligo #217 Length: 000027

AACAACCTCA ATATGGAAGA CCAAGAT

[SEQ. ID NO:270]

Oligo #218 Length: 000018

ATCTTGGTCT TCCATATT

[SEQ. ID NO:271]

Oligo #219 Length: 000027

AACAACCTCA ATTTCGAAGA CCAAGAT

[SEQ. ID NO:272]

Oligo #220 Length: 000018

ATCTTGGTCT TCGAAATT

[SEQ. ID NO:273]

Oligo #221 Length: 000027

AACAACCTCA ATACCGAAGA CCAAGAT

[SEQ. ID NO:274]

Oligo #222 Length: 000018

ATCTTGGTCT TCGGTATT

[SEQ. ID NO:275]

Oligo #223 Length: 000027

AACAACCTCA ATTACGAAGA CCAAGAT

[SEQ. ID. NO:276]

Oligo #224 Length: 000018

ATCTTGGTCT TCGTAATT

[SEQ. ID NO:277]

Oligo #225 Length: 000027

AACAACCTCA ATGTTGAAGA CCAAGAT

[SEQ. ID NO:278]

Oligo #226 Length: 000018

ATCTTGGTCT TCAACATT

[SEQ. ID NO:279]

Oligo #227 Length: 000027

AACAACCTCA ATGGGCGTGA CCAAGAT

[SEQ. ID NO:280]

Oligo #228 Length: 000018

ATCTTGGTCT CGCCCATT

[SEQ. ID NO:281]

Oligo #229 Length: 000027

AACAACCTCA ATGGGCAGGA CCAAGAT

[SEQ. ID NO:282]

Oligo #230 Length: 000018

ATCTTGGTCC TGCCCATT

[SEQ. ID NO:283]

Oligo #231 Length: 000027

AACAACCTCA ATGGGGGTGA CCAAGAT

[SEQ. ID NO:284]

Oligo #232 Length: 000018

ATCTTGGTCA CCCCCATT

[SEQ. ID NO:285]

Oligo #233 Length: 000027

AACAACCTCA ATGGGACCGA CCAAGAT

[SEQ. ID NO:286]

Oligo #234 Length: 000018

ATCTTGGTCG GTCCCATT

[SEQ. ID NO:287]

Oligo #235 Length: 000027

AACAACCTCA ATGGGGAAGC TCAAGAT

[SEQ. ID NO:288]

Oligo #236 Length: 000018

ATCTTGAGCT TCCCCATT

[SEQ. ID NO:289]

Oligo #237 Length: 000027

AACAACCTCA ATGGGGAAAA CCAAGAT

[SEQ. ID NO:290]

Oligo #238 Length: 000018

ATCTTGGTTT TCCCCATT

[SEQ. ID NO:291]

Oligo #239 Length: 000027

AACAACCTCA ATGGGGAACA GCAAGAT

[SEQ. ID NO:292]

Oligo #240 Length: 000018

ATCTTGCTGT TCCCCATT

[SEQ. ID NO:293]

Oligo #241 Length: 000027

AACAACCTCA ATGGGGAAGA ACAAGAT

[SEQ. ID NO:294]

Oligo #242 Length: 000018

ATCTTGTTCT TCCCCATT

[SEQ. ID NO:295]

Oligo #243 Length: 000027

AACAACCTCA ATGGGGAAGA CGCTGAT

[SEQ. ID NO:296]

Oligo #244 Length: 000018

ATCAGCGTCT TCCCCATT

[SEQ. ID NO:297]

Oligo #245 Length: 000027

AACAACCTCA ATGGGGAAGA CCGTGAT

[SEQ. ID NO:298]

Oligo #246 Length: 000018

ATCACGGTCT TCCCCATT

[SEQ. ID NO:299]

Oligo #247 Length: 000027

AACAACCTCA ATGGGGAAGA CAACGAT

[SEQ. ID NO:300]

Oligo #248 Length: 000018

ATCGTTGTCT TCCCCATT

[SEQ. ID NO:301]

Oligo #249 Length: 000027

AACAACCTCA ATGGGGAAGA CGACGAT

[SEQ. ID NO:302]

Oligo #250 Length: 000018

ATCGTCGTCT TCCCCATT

[SEQ. ID NO:303]

Oligo #251 Length: 000027

AACAACCTCA ATGGTGAAGA CGAAGAT

[SEQ. ID NO:304]

Oligo #252 Length: 000018

ATCTTCGTCT TCCCCATT

[SEQ. ID NO:305]

Oligo #253 Length: 000027

AACAACCTCA ATGGTGAAGA CCACGAT

[SEQ. ID NO:306]

Oligo #254 Length: 000018

ATCGTGGTCT TCCCCATT

[SEQ. ID NO:307]

Oligo #255 Length: 000027

AACAACCTCA ATGGGGAAGA CATCGAT

[SEQ. ID NO:308]

Oligo #256 Length: 000018

ATCGATGTCT TCCCCATT

[SEQ. ID NO:309]

Oligo #257 Length: 000027

AACAACCTCA ATGGGGAAGA CTCCGAT

[SEQ. ID NO:310]

Oligo #258 Length: 000018

ATCGGAGTCT TCCCCATT

[SEQ. ID NO:311]

Oligo #259 Length: 000027

AACAACCTCA ATGGGGAAGA CCAAGCT

[SEQ. ID NO:312]

Oligo #260 Length: 000018

AGCTTGGTCT TCCCCATT

[SEQ. ID NO:313]

Oligo #261 Length: 000027

AACAACCTCA ATGGGGAAGA CCAAAAC

[SEQ. ID NO:314]

Oligo #262 Length: 000018

GTTTTGGTCT TCCCCATT

[SEQ. ID NO:315]

Oligo #263 Length: 000027

AACAACCTCA ATGGGGAAGA CCAACAG

[SEQ. ID NO:316]

Oligo #264 Length: 000018

CTGTTGGTCT TCCCCATT

[SEQ. ID NO:317]

Oligo #265 Length: 000027

AACAACCTCA ATGGGGAAGA CCAAGAA

[SEQ. ID NO:318]

Oligo #266 Length: 000018

TTCTTGGTCT TCCCCATT

[SEQ. ID NO:319]

Oligo #267 Length: 000027

AACAACCTCA ATGGGGAAGA CCAACAC

[SEQ. ID NO:320]

Oligo #268 Length: 000018

GTGTTGGTCT TCCCCATT

[SEQ. ID NO:321]

Oligo #269 Length: 000027

AACAACCTCA ATGGGGAAGA CCAAATC

[SEQ. ID NO:322]

Oligo #270 Length: 000018

GATTTGGTCT TCCCCATT

[SEQ. ID NO:323]

Oligo #271 Length: 000027

AACAACCTCA ATGGGGAAGA CCAACTG

[SEQ. ID NO:324]

Oligo #272 Length: 000018

CAGTTGGTCT TCCCCATT

[SEQ. ID NO:325]

Oligo #273 Length: 000027

AACAACCTCA ATGGGGAAGA CCAAAAA

[SEQ. ID NO:326]

Oligo #274 Length: 000018

TTTTTGGTCT TCCCCATT

[SEQ. ID NO:327]

Oligo #275 Length: 000027

AACAACCTCA ATGGGGAAGA CCAATAC

[SEQ. ID NO:328]

Oligo #276 Length: 000018

GTATTGGTCT TCCCCATT

[SEQ. ID NO:329]

Oligo #277 Length: 000027

AACAACCTCA ATGGGGAAGA CCAAGTT

[SEQ. ID NO:330]

Oligo #278 Length: 000018

AACTTGGTCT TCCCCATT

[SEQ. ID NO:331]

Oligo #279 Length: 000036

ATCGCTATGG AAAATAACCT TCGAAGGCCA AACCTG

[SEQ. ID NO:332]

Oligo #280 Length: 000027

CCTTCGAAGG TTATTTTCCA TAGCGAT

[SEQ. ID NO:333]

Oligo #281 Length: 000036

ATCGAAATGG AAAATAACCT TCGAAGGCCA AACCTG

[SEQ. ID NO:334]

Oligo #282 Length: 000027

CCTTCGAAGG TTATTTTCCA TTTCGAT

[SEQ. ID NO:335]

Oligo #283 Length: 000036

ATCAAAATGG AAAATAACCT TCGAAGGCCA AACCTG

[SEQ. ID NO:336]

Oligo #284 Length: 000027

CCTTCGAAGG TTATTTTCCA TTTTGAT

[SEQ. ID NO:337]

Oligo #285 Length: 000036

ATCATGATGG AAAATAACCT TCGAAGGCCA AACCTG

(SEQ. ID NO:338]

Oligo #286 Length: 000027

CCTTCGAAGG TTATTTTCCA TCATGAT

[SEQ. ID NO:339]

Oligo #287 Length: 000036

ATCACCATGG AAAATAACCT TCGAAGGCCA AACCTG

[SEQ. ID NO:340]

Oligo #288 Length: 000027

CCTTCGAAGG TTATTTTCCA TGGTGAT

[SEQ. ID NO:341]

O1igo #289 Length: 000036

ATCGTTATGG AAAATAACCT TCGAAGGCCA AACCTG

[SEQ. ID NO:342]

Oligo #290 Length: 000027

CCTTCGAAGG TTATTTTCCA TAACGAT

[SEQ. ID NO:343]

Oligo #291 Length: 000036

ATCCTGATGC ACAATAACCT TCGAAGGCCA AACCTG

[SEQ. ID NO:344]

Oligo #292 Length: 000027

CCTTCGAAGG TTATTGTGCA TCAGGAT

[SEQ. ID NO:345]

Oligo #293 Length: 000036

ATCCTGATGA TGAATAACCT TCGAAGGCCA AACCTG

[SEQ. ID NO:346]

Oligo #294 Length: 000027

CCTTCGAAGG TTATTCATCA TCAGGAT

[SEQ. ID NO:347]

Oligo #295 Length: 000036

ATCCTGATGT TCAATAACCT TCGAAGGCCA AACCTG

[SEQ. ID NO:348]

Oligo #296 Length: 000027

CCTTCGAAGG TTATTGAACA TCAGGAT

[SEQ. ID NO:349]

Oligo #297 Length: 000036

ATCCTGATGG CTAATAACCT TCGAAGGCCA AACCTG

[SEQ. ID NO:350]

Oligo #298 Length: 000027

CCTTCGAAGG TTATTAGCCA TCAGGAT

[SEQ. ID NO:351]

Oligo #299 Length: 000036

ATCCTGATGA ACAATAACCT TCGAAGGCCA AACCTG

[SEQ. ID NO:352]

Oligo #300 Length: 000027

CCTTCGAAGG TTATTGTTCA TCAGGAT

[SEQ. ID NO:353]

Oligo #301 Length: 000036

ATCCTGATGA TCAATAACCT TCGAAGGCCA AACCTG

[SEQ. ID NO:354]

Oligo #302 Length: 000027

CCTTCGAAGG TTATTGATCA TCAGGAT

[SEQ. ID NO:355]

Oligo #303 Length: 000036

ATCCTGATGA AAAATAACCT TCGAAGGCCA AACCTG

[SEQ. ID NO:356]

Oligo #304 Length: 000027

CCTTCGAAGG TTATTTTTCA TCAGGAT

[SEQ. ID NO:357]

Oligo #305 Length: 000036

ATCCTGATGT CCAATAACCT TCGAAGGCCA AACCTG

[SEQ. ID NO:358]

Oligo #306 Length: 000027

CCTTCGAAGG TTATTGGACA TCAGGAT

[SEQ. ID No:359]

Oligo #307 Length: 000036

ATCCTGATGG TTAATAACCT TCGAAGGCCA AACCTG

[SEQ. ID NO:360]

Oligo #308 Length: 000027

CCTTCGAAGG TTATTAACCA TCAGGAT

[SEQ. ID NO:361]

Oligo #309 Length: 000036

ATCCTGATGG AAAATAACCT TGCTAGGCCA AACCTG

[SEQ. ID NO:362]

Oligo #310 Length: 000027

CCTAGCAAGG TTATTTTCCA TCAGGAT

[SEQ. ID NO:363]

Oligo #311 Length: 000036

ATCCTGATGG AAAATAACCT TAACAGGCCA AACCTG

[SEQ. ID NO:364]

Oligo #312 Length: 000027

CCTGTTAAGG TTATTTTCCA TCAGGAT

[SEQ. ID NO:365]

Oligo #313 Length: 000036

ATCCTGATGG AAAATAACCT TCACAGGCCA AACCTG

[SEQ. ID NO:366]

Oligo #314 Length: 000027

CCTGTGAAGG TTATTTTCCA TCAGGAT

[SEQ. ID NO:367]

Oligo #315 Length: 000036

ATCCTGATGG AAAATAACCT TAAAAGGCCA AACCTG

[SEQ. ID NO:368]

Oligo #316 Length: 000027

CCTTTTAAGG TTATTTTCCA TCAGGAT

[SEQ. ID NO:369]

Oligo #317 Length: 000036

ATCCTGATGG AAAATAACCT TCGAAGGGCT AACCTG

[SEQ. ID NO:370]

Oligo #318 Length: 000024

CCTGTTGAAT GCCTCCAGGT TAGC

[SEQ. ID NO:371]

Oligo #319 Length: 000036

ATCCTGATGG AAAATAACCT TCGAAGGCGT AACCTG

[SEQ. ID NO:372]

Oligo #320 Length: 000024

CCTGTTGAAT GCCTCCAGGT TACG

[SEQ. ID NO:373]

Oligo #321 Length: 000036

ATCCTGATGG AAAATAACCT TCGAAGGAAC AACCTG

[SEQ. ID NO:374]

Oligo #322 Length: 000024

CCTGTTGAAT GCCTCCAGGT TGTT

[SEQ. ID NO:375]

Oligo #323 Length: 000036

ATCCTGATGG AAAATAACCT TCGAAGGGAA AACCTG

[SEQ. ID NO:376]

Oligo #324 Length: 000024

CCTGTTGAAT GCCTCCAGGT TTTC

[SEQ. ID NO:377]

Oligo #325 Length: 000036

ATCCTGATGG AAAATAACCT TCGAAGGCAC AACCTG

[SEQ. ID NO:378]

Oligo #326 Length: 000024

CCTGTTGAAT GCCTCCAGGT TGTG

[SEQ. ID NO:379]

Oligo #327 Length: 000036

ATCCTGATGG AAAATAACCT TCGAAGGCTG AACCTG

[SEQ. ID NO:380]

Oligo #328 Length: 000024

CCTGTTGAAT GCCTCCAGGT TCAG

[SEQ. ID NO:381]

Oligo #329 Length: 000036

ATCCTGATGG AAAATAACCT TCGAAGGTTC AACCTG

[SEQ. ID NO:382]

Oligo #330 Length: 000024

CCTGTTGAAT GCCTCCAGGT TGAA

[SEQ. ID NO:383]

Oligo #331 Length: 000036

ATCCTGATGG AAAATAACCT TCGAAGGACC AACCTG

[SEQ. ID NO:384]

Oligo #332 Length: 000024

CCTGTTGAAT GCCTCCAGGT TGGT

[SEQ. ID NO:385]

Oligo #333 Length: 000036

ATCCTGATGG AAAATAACCT TCGAAGGTAC AACCTG

[SEQ. ID NO:386]

Oligo #334 Length: 000024

CCTGTTGAAT GCCTCCAGGT TGTA

[SEQ. ID NO:387]

Oligo #335 Length: 000036

ATCCTGATGG AAAATAACCT TCGAAGGGTT AACCTG

[SEQ. ID NO:388]

Oligo #336 Length: 000024

CCTGTTGAAT GCCTCCAGGT TAAC

[SEQ. ID NO:389]

Oligo #337 Length: 000018

AAAAATCTCG CTCCATGT

[SEQ. ID NO:390]

Oligo #338 Length: 000016

AGCGAGATTT TTAAGAAT

[SEQ. ID NO:391]

Oligo #339 Length: 000018

AAAAATCTCA ACCCATGT

[SEQ. ID NO:392]

Oligo #340 Length: 000018

GTTGAGATTT TTAAGAAT

[SEQ. ID NO:393]

Oligo #341 Length: 000018

AAAAATCTCG AACCATGT

[SEQ. ID NO:394]

Oligo #342 Length: 000018

TTCGAGATTT TTAAGAAT

[SEQ. ID NO:395]

Oligo #343 Length: 000018

AAAAATCTCC ACCCATGT

[SEQ. ID NO:396]

Oligo #344 Length: 000018

GTGGAGATTT TTAAGAAT

[SEQ. ID NO:397]

Oligo #345 Length: 000018

AAAAATCTCA TCCCATGT

[SEQ. ID NO:398]

Oligo #346 Length: 000018

GATGAGATTT TTAAGAAT

[SEQ. ID NO:399]

Oligo #347 Length: 000018

AAAAATCTCA TGCCATGT

[SEQ. ID NO:400]

Oligo #348 Length: 000018

CATGAGATTT TTAAGAAT

[SEQ. ID NO:401]

Oligo #349 Length: 000018

AAAAATCTCT TCCCATGT

[SEQ. ID NO:402]

Oligo #350 Length: 000018

GAAGAGATTT TTAAGAAT

[SEQ. ID NO:403]

Oligo #351 Length: 000018

AAAAATCTCT CCCCATGT

[SEQ. ID NO:404]

Oligo #352 Length: 000018

GGAGAGATTT TTAAGAAT

[SEQ. ID NO:405]

Oligo #353 Length: 000018

AAAAATCTCA CCCCATGT

[SEQ. ID NO:406]

Oligo #354 Length: 000018

GGTGAGATTT TTAAGAAT

[SEQ. ID NO:407]

Oligo #355 Length: 000018

AAAAATCTCT ACCCATGT

[SEQ. ID NO:408]

Oligo #356 Length: 000018

GTAGAGATTT TTAAGAAT

[SEQ. ID NO:409]

Oligo #357 Length: 000027

CTGCCCCTGG CCACGGCCGC AGCTACG

[SEQ. ID NO:410]

Oligo #358 Length: 000024

ATGGATTGGA TGTCGCGTAG CTGC

[SEQ. ID NO:411]

Oligo #359 Length: 000027

CTGCCCCTGG CCACGGCCGC AGGTACG

[SEQ. ID NO:412]

Oligo #360 Length: 000024

ATGGATTGGA TGTCGCGTAC CTGC

[SEQ. ID NO:413]

Oligo #361 Length: 000027

CTGCCCCTGG CCACGGCCGC AATCACG

[SEQ. ID NO:414]

Oligo #362 Length: 000024

ATGGATTGGA TGTCGCGTGA TTGC

[SEQ. ID NO:415]

Oligo #363 Length: 000021

GCTCATCCAA TCCATATCAA G

[SEQ. ID NO:416]

Oligo #364 Length: 000024

ATGGATTGGA TGAGCCGTGG GTGC

[SEQ. ID NO:417]

Oligo #365 Length: 000021

CAGCATCCAA TCCATATCAA G

[SEQ. ID NO:418]

Oligo #366 Length: 000024

ATGGATTGGA TGCTGCGTGG GTGC

[SEQ. ID NO:419]

Oligo #367 Length: 000021

CACCATCCAA TCCATATCAA G

[SEQ. ID NO:420]

Oligo #368 Length: 000024

ATGGATTGGA TGGTGCGTGG GTGC

[SEQ. ID NO:421]

Oligo #369 Length: 000021

AAACATCCAA TCCATATCAA G

[SEQ. ID NO:422]

Oligo #370 Length: 000024

ATGGATTGGA TGTTTCGTGG GTGC

[SEQ. ID NO:423]

Oligo #371 Length: 000021

CGAGCTCCAA TCCATATCAA G

[SEQ. ID NO:424]

Oligo #372 Length: 000024

ATGGATTGGA GCTCGCGTGG GTGC

[SEQ. ID NO:425]

Oligo #373 Length: 000021

CGAAACCCAA TCCATATCAA G

[SEQ. ID NO:426]

Oligo #374 Length: 000024

ATGGATTGGG TTTCGCGTGG GTGC

[SEQ. ID NO:427]

Oligo #375 Length: 000021

CGAGACCCAA TCCATATCAA G

[SEQ. ID NO:428]

Oligo #376 Length: 000024

ATGGATTGGG TCTCGCGTGG GTGC

[SEQ. ID NO:429]

Oligo #377 Length: 000021

CGAATCCCAA TCCATATCAA G

[SEQ. ID NO:430]

Oligo #378 Length: 000024

ATGGATTGGG ATTCGCGTGG GTGC

[SEQ. ID NO:431]

Oligo #379 Length: 000021

CGAAAACCAA TCCATATCAA G

[SEQ. ID NO:432]

Oligo #380 Length: 000024

ATGGATTGGT TTTCGCGTGG GTGC

[SEQ. ID NO:433]

Oligo #381 Length: 000021

CGAATGCCAA TCCATATCAA G

[SEQ. ID NO:434]

Oligo #382 Length: 000024

ATGGATTGGC ATTCGCGTGG GTGC

[SEQ. ID NO:435]

Oligo #383 Length: 000021

CGATTCCCAA TCCATATCAA G

[SEQ. ID NO:436]

Oligo #384 Length: 000024

ATGGATTGGG AATCGCGTGG GTGC

[SEQ. ID NO:437]

Oligo #385 Length: 000021

CGATCCCCAA TCCATATCAA G

[SEQ. ID NO:438]

Oligo #386 Length: 000024

ATGGATTGGG GATCGCGTGG GTGC

[SEQ. ID NO:439]

Oligo #387 Length: 000021

CGATGGCCAA TCCATATCAA G

[SEQ. ID NO:440]

Oligo #388 Length: 000024

ATGGATTGGC CATCGCGTGG GTGC

[SEQ. ID NO:441]

Oligo #389 Length: 000021

CGATACCCAA TCCATATCAA G

[SEQ. ID NO:442]

Oligo #390 Length: 000024

ATGGATTGGG TATCGCGTGG GTGC

[SEQ. ID NO:443]

Oligo #391 Length: 000034

CATCCAATCC AAATCAAGGA CGGTGACTGG AATG

[SEQ. ID NO:444]

Oligo #392 Length: 000044

AATTCATTCC AGTCACCGTC CTTGATTTGG ATTGGATGTC GCGT

[SEQ. ID NO:445]

Oligo #393 Length: 000034

CATCCAATCG AAATCAAGGA CGGTGACTGG AATG

[SEQ. ID NO:446]

Oligo #394 Length: 000044

AATTCATTCC AGTCACCGTC CTTGATTTCG ATTGGATGTC GCGT

[SEQ. ID NO:447]

Oligo #395 Length: 000034

CATCCAATCA TGATCAAGGA CGGTGACTGG AATG

[SEQ. ID NO:448]

Oligo #396 Length: 000044

AATTCATTCC AGTCACCGTC CTTGATCATG ATTGGATGTC GCGT

[SEQ. ID NO:449]

Oligo #397 Length: 000034

CATCCAATCT TCATCAAGGA CGGTGACTGG AATG

[SEQ. ID NO:450]

Oligo #398 Length: 000044

AATTCATTCC AGTCACCGTC CTTGATGAAG ATTGGATGTC GCGT

[SEQ. ID No:451]

Oligo #399 Length: 000034

CATCCAATCT CCATCAAGGA CGGTGACTGG AATG

[SEQ. ID NO:452]

Oligo #400 Length: 000044

AATTCATTCC AGTCACCGTC CTTGATGGAG ATTGGATGTC GCGT

[SEQ. ID NO:453]

Oligo #401 Length: 000034

CATCCAATCg taATCAAGGA CGGTGACTGG AATG

[SEQ. ID NO:454]

Oligo #402 Length: 000044

AATTCATTCC AGTCACCGTC CTTGATTACG ATTGGATGTC GCGT

[SEQ. ID NO:455]

Oligo #403 Length: 000021

CGACATCCAA TCCGTATCAA G

[SEQ. ID NO:456]

Oligo #404 Length: 00024

ACGGATTGGA TGTCGCGTGG GTGC

[SEQ. ID NO:457]

Oligo #405 Length: 000021

CGACATCCAA TCAAAATCAA G

[SEQ. ID NO:458]

Oligo #406 Length: 000024

TTTGATTGGA TGTCGCGTGG GTGC

[SEQ. ID NO:459]

Oligo #407 Length: 000021

CGACATCCAA TCTACATCAA G

[SEQ. ID NO:460]

Oligo #408 Length: 000024

GTAGATTGGA TGTCGCGTGG GTGC

[SEQ. ID NO:461]

Oligo #409 Length: 000016

GCTGGTGACT GGAATG

[SEQ. ID NO:462]

Oligo #410 Length: 000026

AATTCATTCC AGTCACCAGC CTTGAT

[SEQ. ID NO.463]

Oligo #411 Length: 000016

AACGGTGACT GGAATG

[SEQ. ID NO:464]

Oligo #412 Length: 000026

AATTCATTCC AGTCACCGTT CTTGAT

[SEQ. ID NO:465]

Oligo #413 Length: 000016

GAAGGTGACT GGAATG

[SEQ. ID NO:466]

Oligo #414 Length: 000026

AATTCATTCC AGTCACCTTC CTTGAT

[SEQ. ID NO:467]

Oligo #415 Length: 000016

GGTGGTGACT GGAATG

[SEQ. ID NO:468]

Oligo #416 Length: 000026

AATTCATTCC AGTCACCACC CTTGAT

[SEQ. ID NO:469]

Oligo #417 Length: 000016

ATCGGTGACT GGAATG

[SEQ. ID NO:470]

Oligo #418 Length: 000026

AATTCATTCC AGTCACCGAT CTTGAT

[SEQ. ID NO:471]

Oligo #419 Length: 000016

CTGGGTGACT GGAATG

[SEQ. ID NO:472]

Oligo #420 Length: 000026

AATTCATTCC AGTCACCCAG CTTGAT

[SEQ. ID NO:473]

Oligo #421 Length: 000016

TTCGGTGACT GGAATG

[SEQ. ID NO:474]

Oligo #422 Length: 000026

AATTCATTCC AGTCACCGAA CTTGAT

[SEQ. ID NO:475]

Oligo #423 Length: 000016

TCCGGTGACT GGAATG

[SEQ. ID NO:476]

Oligo #424 Length: 000026

AATTCATTCC AGTCACCGGA CTTGAT

[SEQ. ID NO:477]

Oligo #425 Length: 000032

AATTCGCTAG GAAACTGACG TTCTATCTGA AA

[SEQ. ID NO:478]

Oligo #426 Length: 000037

CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTAGCG

[SEQ. ID NO:479]

Oligo #427 Length: 000032

AATTCCAGAG GAAACTGACG TTCTATCTGA AA

[SEQ. ID NO:480]

Oligo #428 Length: 000037

CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTCTGG

[SEQ. ID NO:481]

Oligo #429 Length: 000032

AATTCCACAG GAAACTGACG TTCTATCTGA AA

[SEQ. ID NO:482]

Oligo #430 Length: 000037

CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTGTGG

[SEQ. ID NO:483]

Oligo #431 Length: 000032

AATTCTCCAG GAAACTGACG TTCTATCTGA AA

[SEQ. ID NO:484]

Oligo #432 Length: 000037

CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTGGAG

[SEQ. ID NO:485]

Oligo #433 Length: 000032

AATTCCGGAG GCGTCTGACG TTCTATCTGA AA

[SEQ. ID NO:486]

Oligo #434 Length: 000037

CTCAAGGGTT TTCAGATAGA ACGTCAGACG CCTCCGG

[SEQ. ID NO:487]

Oligo #435 Length: 000032

AATTCCGGAG GGAACTGACG TTCTATCTGA AA

[SEQ. ID NO:488]

Oligo #436 Length: 000037

CTCAAGGGTT TTCAGATAGA ACGTCAGTTC CCTCCGG

[SEQ. ID NO:489]

Oligo #437 Length: 000032

AATTCCGGAG GCACCTGACG TTCTATCTGA AA

[SEQ. ID NO:490]

Oligo #438 Length: 000037

CTCAAGGGTT TTCAGATAGA ACGTCAGGTG CCTCCGG

[SEQ. ID NO:491]

Oligo #439 Length: 000032

AATTCCGGAG GATCCTGACG TTCTATCTGA AA

[SEQ. ID NO:492]

Oligo #440 Length: 000037

CTCAAGGGTT TTCAGATAGA ACGTCAGGAT CCTCCGG

[SEQ. ID NO:493]

Oligo #441 Length: 000032

AATTCCGGAG GTCCCTGACG TTCTATCTGA AA

[SEQ. ID NO:494]

Oligo #442 Length: 000037

CTCAAGGGTT TTCAGATAGA ACGTCAGGGA CCTCCGG

[SEQ. ID NO:495]

Oligo #443 Length: 000032

AATTCCGGAG GAAACTGACG GACTATCTGA AA

[SEQ. ID NO:496]

Oligo #444 Length: 000037

CTCAAGGGTT TTCAGATAGT CCGTCAGTTT CCTCCGG

[SEQ. ID NO:497]

Oligo #445 Length: 000032

AATTCCGGAG GAAACTGACG ATCTATCTGA AA

[SEQ. ID NO:498]

Oligo #446 Length: 000037

CTCAAGGGTT TTCAGATAGA TCGTCAGTTT CCTCCGG

[SEQ. ID NO:499]

Oligo #447 Length: 000032

AATTCCGGAG GAAACTGACG CTGTATCTGA AA

[SEQ. ID NO:500]

Oligo #448 Length: 000037

CTCAAGGGTT TTCAGATACA GCGTCAGTTT CCTCCGG

[SEQ. ID NO:501]

Oligo #449 Length: 000032

AATTCCGGAG GAAACTGACG AAATATCTGA AA

[SEQ. ID NO:502]

Oligo #450 Length: 000037

CTCAAGGGTT TTCAGATATT TCGTCAGTTT CCTCCGG

[SEQ. ID NO:503]

Oligo #451 Length: 000032

AATTCCGGAG GAAACTGACG GTTTATCTGA AA

[SEQ. ID NO:504]

Oligo #452 Length: 000037

CTCAAGGGTT TTCAGATAAA CCGTCAGTTT CCTCCGG

[SEQ. ID NO:505]

Oligo #453 Length: 000032

AATTCCGGAG GAAACTGACG TTCTATCTGG CT

[SEQ. ID NO:506]

Oligo #454 Length: 000037

CTCAAGGGTA GCCAGATAGA ACGTCAGTTT CCTCCGG

[SEQ. ID NO:507]

Oligo #455 Length: 000032

AATTCCGGAG GAAACTGACG TTCTATCTGC GT

[SEQ. ID NO:508]

Oligo #456 Length: 000037

CTCAAGGGTA CGCAGATAGA ACGTCAGTTT CCTCCGG

[SEQ. ID NO:509]

Oligo #457 Length: 000032

AATTCCGGAG GAAACTGACG TTCTATCTGA AC

[SEQ. ID NO:510]

Oligo #458 Length: 000037

CTCAAGGGTG TTCAGATAGA ACGTCAGTTT CCTCCGG

[SEQ. ID NO:511]

Oligo #459 Length: 000032

AATTCCGGAG GAAACTGACG TTCTATCTGC AG

[SEQ. ID NO:512]

Oligo #460 Length: 000037

CTCAAGGGTC TGCAGATAGA ACGTCAGTTT CCTCCGG

[SEQ. ID NO:513]

Oligo #461 Length: 000032

AATTCCGGAG GAAACTGACG TTCTATCTGC AC

[SEQ. ID NO:514]

Oligo #462 Length: 000037

CTCAAGGGTG TGCAGATAGA ACGTCAGTTT CCTCCGG

[SEQ. ID NO:515]

Oligo #463 Length: 000032

AATTCCGGAG GAAACTGACG TTCTATCTGA TG

[SEQ. ID NO:516]

Oligo #464 Length: 000037

CTCAAGGGTC ATCAGATAGA ACGTCAGTTT CCTCCGG

[SEQ. ID NO:517]

Oligo #465 Length: 000032

AATTCCGGAG GAAACTGACG TTCTATCTGT TC

[SEQ. ID NO:518]

Oligo #466 Length: 000037

CTCAAGGGTG AACAGATAGA ACGTCAGTTT CCTCCGG

[SEQ. ID NO:519]

Oligo #467 Length: 000032

AATTCCGGAG GAAACTGACG TTCTATCTGT AC

[SEQ. ID NO:520]

Oligo #468 Length: 000037

CTCAAGGGTG TACAGATAGA ACGTCAGTTT CCTCCGG

[SEQ. ID NO:521]

Oligo #469 Length: 000040

CATGGCTAAC TGCTCTAACA TGATCGATGA AATTATAACA

[SEQ. ID NO:522]

Oligo #470 Length: 000036

CACTTAAAGC AGCCACCTTT GCCTTTGCTG GACTTC

[SEQ. ID NO:523]

Oligo #471 Length: 000027

AACAACCTCA ATGGGGAAGA CCAAGAT

[SEQ. ID NO:524]

Oligo #472 Length: 000045

CTTTAAGTGT GTTATAATTT CATCGATCAT GTTAGAGCAG TTAGC

[SEQ. ID NO:525]

Oligo #473 Length: 000036

GAGGTTGTTG AAGTCCAGCA AAGGCAAAGG TGGCTG

[SEQ. ID NO:526]

Oligo #474 Length: 000018

ATCTTGGTCT TCCCCATT

[SEQ. ID NO:527]

Oligo #475 Length: 000036

ATCCTGATGG AAAATAACCT TCGAAGGCCA AACCTG

[SEQ. ID NO:528]

Oligo #476 Length: 000024

GAGGCATTCA ACAGGGCTGT CAAG

[SEQ. ID NO:529]

Oligo #477 Length: 000015

AGTTTACAGA ATGCA

[SEQ. ID NO:530]

Oligo #478 Length: 000027

CCTTCGAAGG TTATTTTCCA TCAGGAT

[SEQ. ID NO:531]

Oligo #479 Length: 000024

CCTGTTGAAT GCCTCCAGGT TTGG

[SEQ. ID NO:532]

Oligo #480 Length: 000020

TTCTGTAAAC TCTTGACAGC

[SEQ. ID NO:533]

Oligo #481 Length: 000021

TCAGCAATTG AGAGCATTCT T

[SEQ. ID NO:534]

Oligo #482 Length: 000018

AAAAATCTCC TGCCATGT

[SEQ. ID NO:535]

Oligo #483 Length: 000048

CTGCCCCTGG CCACGGCCGC ACCCACGCGA CATCCAATCC ATATCAAG

[SEQ. ID NO:536]

Oligo #484 Length: 000027

CTGCCCCTGG CCACGGCCGC ACCCACG

[SEQ. ID NO:537]

Oligo #485 Length: 000021

CGACATCCAA TCCATATCAA G

[SEQ. ID NO:538]

Oligo #486 Length: 000016

GACGGTGACT GGAATG

[SEQ. ID NO:539]

Oligo #487 Length: 000019

GCTCTCAATT GCTGATGCA

[SEQ. ID NO:540]

Oligo #488 Length: 000018

CAGGAGATTT TTAAGAAT

[SEQ. ID NO:541]

Oligo #489 Length: 000048

ATGGATTGGA TGTCGCGTGG GTGCGGCCGT GGCCAGGGGC AGACATGG

[SEQ. ID NO:542]

Oligo #490 Length: 000024

GGCCGTGGCC AGGGGCAGAC ATGG

[SEQ. ID NO:543]

0ligo #491 Length: 000024

ATGGATTGGA TGTCGCGTGG GTGC

[SEQ. ID NO:544]

Oligo #492 Length: 000026

AATTCATTCC AGTCACCGTC CTTGAT

[SEQ. ID NO:545]

Oligo #493 Length: 000032

AATTCCGGAG GAAACTGACG TTCTATCTGA AA

[SEQ. ID NO:546]

Oligo #494 Length: 000032

ACCCTTGAGA ATGCGCAGGC TCAACAGTAA TA

[SEQ. ID NO:547]

Oligo #495 Length: 000037

CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTCCGG

[SEQ. ID NO:548]

Oligo #496 Length: 000027

AGCTTATTAC TGTTGAGCCT GCGCATT

[SEQ. ID NO:549]

TABLE 3

POLYPEPTIDES

PEPTIDE #1; pMON5988 (Examp1e 9); (15-125)hIL-3

Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu

[SEQ ID NO:65]

15 20 25

Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly

30 35 40

Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn

45 50 55

Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser

60 65 70

Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu PrO Cys Leu Pro Leu

75 80 85

Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly

90 95 100

Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr

105 110 115

Leu Glu Asn Ala Gln Ala Gln Gln

120 125

PEPTIDE #A1; pMON13304 (Example 55); Met-Ala-(15-125)hIL-3

(98I, 100R):

Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu

[SEQ ID NO:66]

15 20 25

Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly

30 35 40

Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn

45 50 55

Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser

60 65 70

Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu

75 80 85

Ala Thr Ala Ala Pro Thr Arg His Pro Ile Ile Ile Arg Asp Gly

90 95 100

Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr

105 110 115

Leu Glu Asn Ala Gln Ala Gln Gln

120 125

PEPTIDE #A2; pMON133O5 Met-Ala-(15-125)hIL-3;

(95R, 98I, 100R);

Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile IIe Thr His Leu

[SEQ ID NO:67]

15 20 25

Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly

30 35 40

Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn

45 50 55

Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser

60 65 70

Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu

75 80 85

Ala Thr Ala Ala Pro Thr Arg Arg Pro Ile Ile Ile Arg Asp Gly

90 95 100

Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr

105 110 115

Leu Glu Asn Ala Gln Ala Gln Gln

120 125

PEPTIDE #A3; pMON13286 Met-Ala-(15-125)hIL-3;

(42D, 45M, 46S);

Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu

[SEQ ID NO:69]

15 20 25

Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Asp

30 35 40

Glu Asp Met Ser Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn

45 50 55

Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser

60 65 70

Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu

75 80 85

Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly

90 95 100

Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr

105 110 115

Leu Glu Asn Ala Gln Ala Gln Gln

120 125

Polypeptides corresponding to SEQ ID NOS. 15, 16, 17, 18 and 129 comprising (1-133) hIL-3 containing one or more amino acid substitutions can be made using the procedures described above and in the following examples by starting with the appropriate oligonuctiotides and then constructing the DNA encoding the polypeptide and expressing it in an appropriate host cell. In a similar manner polypeptides which correspond to SEQ ID NOS. 19, 20, 21, 22 and 130 and contain one or more amino acid substitutions and wherein from 1 to 14 amino acids have been sequentially deleted from the N-terminus, or from 1 to 15 amino acids have been deleted from the C-terminus or deletions of amino acids have been made from both the N-terminus and the C-terminus can also be made by following the procedures described above and in the following examples, beginning with the appropriate starting materials.

Additional details may be found in U.S. patent application Ser. No. 07/981,044 filed Nov. 24, 1992, now abandoned which is hereby incorporated by reference in its entirety.

Additional details may be found in co filed U.S. patent application Ser. No. 08/411,795 which is hereby incorporated by reference in its entirety.

All references, patents or applications cited herein are incorporated by reference in their entirety.

Further details known to those skilled in the art may be found in T. Maniatis, et al.,

Molecular Cloning, A Laboratory Manual,

Cold Spring Harbor Laboratory (1982) and references cited therein, incorporated herein by reference in its entirety; and in J. Sambrook, et al.,

Molecular Cloning, A Laboratory Manual,

2nd edition, Cold Spring Harbor Laboratory (1989) and references cited therein, incorporated herein by reference in its entirety.

The following examples will illustrate the invention in greater detail although it will be understood that the invention is not limited to these specific examples.

Amino acids are shown herein by standard one letter or three letter abbreviations as follows:

Abbreviated Designation

Amino Acid

A

Ala

Alanine

C

Cys

Cysteine

D

Asp

Aspartic acid

E

Glu

Glutamic acid

F

Phe

Phenylalanine

G

Gly

Glycine

H

His

Histidine

I

Ile

Isoleucine

K

Lys

Lysine

L

Leu

Leucine

M

Met

Methionine

N

Asn

Asparagine

P

Pro

Proline

Q

Gln

Glutamine

R

Arg

Arginine

S

Ser

Serine

T

Thr

Threonine

V

Val

Valine

W

Trp

Tryptophan

Y

Tyr

Tyrosine

Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention. It is intended that all such other examples be included within the scope of the appended claims.

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EXAMPLE 1

Construction of pMON 5846 (

FIG. 4

) which Encodes [Met-(1-133) hIL-3 (Arg129)]

A plasmid containing the gene for the cDNA of hIL-3 cloned into pUC18 on an EcoRI to HindIII fragment was obtained from British Biotechnology Limited (Cambridge, England). This plasmid was designated pPO518. The purified plasmid DNA was cleaved by the restriction endonucleases NheI and BamHI. Approximately 0.5 micrograms of cleaved plasmid DNA was ligated to 1.0 picomoles of a pair of annealed oligonucleotides with the following sequence:

5′-CTAGCGATCTTTTAATAAGCTTG-3′ [SEQ ID NO: 1]

3′-GCTAGAAAATTATTCGAACCTAG-5′ [SEQ ID NO: 2]

The ligation mixture was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked, and plasmid DNA was purified and subjected to restriction enzyme analysis. An isolate was identified in which the above oligonucleotide sequence had replaced the portion of the gene that encodes the extreme C-terminus. Within the new sequence was a new stop codon, TAA, and a recognition site for the enzyme HindIII. The new plasmid was designated pMON5846.

EXAMPLE 2

(a) Construction of Expression Vector Plasmid pMON2341

The plasmid pMON2341 was used to supply the particular replicon and expression elements used for construction of many of the plasmids used to produce hIL-3 and hIL-3 muteins in

E. coli.

These expression elements are described in the materials and methods section. pMON2341 is derived from pMON5515 (Olins et al., 1988) and from pMON2429. pMON2429 consists of the phage mp18 (Yanisch-Perron et al., 1985) with a BclI fragment carrying the chloramphenicol acetyl transferase (cat) gene from pBR328 (Covarrubias et al., 1981) inserted into the BamHI site. The cat gene in pMON2429 has been altered from that in pBR328 by site directed mutagenesis (Kunkel, 1985). The recognition sites for NcoI and EcoRI which occur in the native gene were altered so that these two restriction enzymes no longer recognize these sites. The changes did not alter the protein specified by the gene. Also, an NcoI site was introduced at the N-terminus of the coding sequence so that it overlaps the codon for initiator methionine.

The steps involved in construction of pMON2341 are listed below:

(1) The DNAs of pMON5515 and pMON2429 were treated with NcoI and HindIII. The fragments were ligated and used to transform competent

E. coli

to ampicillin resistance. From these colonies, some were identified that were chloramphenicol resistant. From one of these colonies, plasmid DNA was isolated in which the rat atriopeptigen gene of pMON5515 had been replaced by the NcoI to HindIII fragment containing the cat gene from pMON2429. This fragment contains the recognition sites for several restriction enzymes in the portion derived, from the multilinker region of mp18. The new plasmid was designated pMON2412.

(2) pMON2412 was treated with the enzyme ClaI which cleaves at one location in the pBR327 derived portion of the DNA. The protruding ends were rendered blunt by treatment with Klenow in the presence of nucleotide precursors. This DNA was mixed with an isolated 514 bp RsaI fragment derived from pEMBL8 (Dente et al., 1983). This RsaI fragment contains the origin of replication of phage f1. This ligation mixture was used to transform competent

E. coli

cells to ampicillin resistance. Among the plasmid DNAs isolated from these cells was pMON5578. This plasmid has the structure of pMON2412 with the f1 origin region inserted into the ClaI site. This in illustrated in the Figures and in Olins and Rangwala (1990).

(3) The DNA of pMON5578 was treated with restriction enzymes HindIII and MstII. The DNA was then treated with Klenow enzyme in the presence of nucleotide precursors to render the ends blunt. This treated DNA was ligated and used to transform competent

E. coli

to ampicillin resistance. From the ampicillin resistant colonies, one plasmid was recovered from which the portion between HindIII and MstII was absent. This deletion resulted in the removal of sequences from the plasmid which are recognized by a number of restriction endonuclease sites. The new plasmid was designated pMON5582.

(4) The DNA of pMON5582 was treated with SstII and BclI and ligated in the presence of annealed oligonucleotides with the sequences shown below.

5′-GGCAACAATTTCTACAAAACACTTGATACTGTATGAGCAT-3′-CGCCGTTGTTAAAGATGTTTTGTGAACTATGACATACTCGTAACAGTATAATTGCTTCAACAGAACAGATC-3′ [SEQ ID NO:3]

TGTCATATTAACGAAGTTGTCTTGT-5′ [SEQ ID NO:4]

This sequence encodes the essential elements of the recA promoter of

E. coli

including the transcription start site and the lexA repressor binding site (the operator) (Sancar et al., 1980). The plasmid recovered from the ligation mixes contained this recA promoter in place of the one In pMON5582 (and in pMON5515). The functionality of the recA promoter was illustrated by Olins and Rangwala (1990). The new plasmid was designated pMON5594.

(5) To eliminate the single EcoRI site in pMON5594, the DNA was treated with EcoRI, then with Klenow in the presence of nucleotide precursors to render the ends blunt and then the DNA was ligated. From this ligation mix a plasmid was recovered whose DNA was not cleaved with EcoRI. This plasmid was designated pMON5630.

(6) To alter the single recognition site for PstI, plasmid pMON5630 was subjected to site directed mutagenesis (Kunkel, 1985). The oligonucleotide used in this procedure has the sequence shown below.

5′-CCATTGCTGCCGGCATCGTGGTC-3′ [SEQ ID NO:5]

The result of the procedure was to construct pMON2341 which differs from pMON5630 in that the PstI site in the beta-lactamase gene was altered so that PstI no longer recognizes the site. The single nucleotide change does not alter the amino acid sequence of the beta-lactamase protein.

(b) Construction of pMON5847 (

FIG. 5

) which Encodes [Met-(1-133) hIL-3(Arg

129

)]

Plasmid pMON2341 was used to supply the replicon, promotor, ribosome binding site, transcription terminator and antibiotic resistance marker for the plasmids used to produce hIL-3 in

E. coli

from cDNA derived hIL-3 genes.

Plasmid pMON2341 was treated with restriction enzymes NcoI and HindIII. The restriction fragment containing the replication origin was purified. The DNA of plasmid pMON5846 was treated with NcoI and HindIII. The restriction fragment containing the hIL-3 gene was gel purified. These purified restriction fragments were mixed and ligated. The ligation mixture was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked, and plasmid DNA was purified and analyzed using restriction enzymes. pMON5847 was identified as a plasmid with the replicon of pMON2341 and the hIL-3 gene in place of the chloramphenicol acetyl transferase gene. JM101 cells harboring this plasmid were cultured in M9 medium and treated with nalidixic acid as described above. Samples of the culture were examined for protein content. It was found that this hIL-3 mutein was produced at about 6% of total cell protein as measured on Coomassie stained polyacrylamide gels.

EXAMPLE 3

Construction of pMON5854 (

FIG. 7

) which Encodes [Met-(1-133) hIL-3(Arg

129

)]

To increase the accumulation of hIL-3 in

E. coli,

the coding sequence of the amino terminal portion of the protein was altered to more closely reflect the codon bias found in

E. coli

genes that produce high levels of proteins (Gouy and Gautier, 1982). To change the coding sequence for the amino terminal portion of the gene, a pair of synthetic oligonucleotides were inserted between the NcoI and HpaI sites within the coding sequence. About 0.5 micrograms of DNA of the plasmid pMON5847 (Example 2) was treated with NcoI and HpaI. This DNA was mixed with an annealed pair of oligonucleotides with the following sequence:

5′-CATGGCTCCAATGACTCAGACTACTTCTCTTAAGACT-3′-CGAGGTTACTGAGTCTGATGAAGAGAATTCTGATCTTGGGTT-3′ [SEQ ID NO:6]

AGAACCCAA-5′ [SEQ ID NO:7]

The fragments were ligated. The ligation mixture was used to transform competent JM101 to ampicillin resistance. Colonies were picked into broth. From the cultures plasmid DNA was made and examined for the presence of a DdeI site (CTNAG) which occurs in the synthetic sequence but not between the NcoI and HpaI sites in the sequence of pMON5847. The new recombinant plasmid was designated pMON5854. The nucleotide sequence of the DNA in the coding sequence of the amino terminal portion of the hIL-3 gene in pMON5854 was determined by DNA sequencing and found to be the same as that of the synthetic oligonucleotide used in ligation. Cultures of JM101 cells harboring this plasmid were grown and treated with nalidixic acid to induce production of the hIL-3 mutant protein. Analysis of the proteins on Coomassie gels showed that the accumulation of hIL-3 mutein was about 25% of total cell protein in cultures harboring pMON5854, significantly higher than it was in cultures harboring pMON5847.

EXAMPLE 4

Construction of pMON5887 (

FIG. 12

) which Encodes [Met-(1-125) hIL-3]

The plasmid DNA of pMON5854 (Example 3) was treated with EcoRI and HindIII and the larger fragment was gel purified. About 0.5 mitrogram of this DNA was ligated to 1 picomole of an annealed pair of oligonucleotides which encode amino acids 107 through 125 of hIL-3. The sequences of these oligonucleotides are shown below. EcoRI to HindIII

5′-AATTCCGTCOTAAMCTGACCTTCTATCTGAAAA-3′-GGCAGCATTTGACTGGAAGATAGACTTTTCCTTGGAGAACGCGCAGGCTCAACAGTAATA-3′ [SEQ ID NO;8]

GGAACCTCTTGCGCGTCCGAGTTGTCATTATTCGA-5′ [SEQ ID NO:9]

After ligation, the DNA was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked into broth and plasmid DNA was isolated from each culture. Restriction analysis of the plasmid DNA showed the presence of an EcoRI to HindIII fragment smaller than that of pMON5854. The nucleotide sequence of the portion of the coding sequence between the EcoRI and HindIII sites was determined to confirm the accuracy of the replaced sequence. The new plasmid was designated pMON5887 encoding Met-(1-125) hIL-3 which has the following amino acid sequence:

[SEQ ID NO:10]

Met Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr

Ser Trp Val Asn Cys Ser Asn Met Ile Asp Glu Ile

Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu

Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile

Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu

Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala

Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro

Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His

Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe

Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu

Asn Ala Gln Ala Gln Gln

EXAMPLE 5

Construction of pMON5967 which Encodes [Met-Ala-(15-125) hIL-3]

Plasmid DNA of pMON5887 isolated from

E. coli

GM48 (dam-) was cleaved with NcoI and ClaI and ligated to 1 picomole of an annealed pair of oligonucleotides, Nco I and ClaI, encoding amino acids [Met Ala (15-20) hIL-3]. The sequence of these oligonucleotides is shown below.

5′-CATGGCTAACTGCTCTAACATGAT-3′[SEQ ID NO:11]

3′-CGATTGACGAGATTGTACTAGC-5′[SEQ ID NO:12]

The resulting ligation mix was used to transform competent

E. coli

JM101 cells to ampicillin resistant colonies. Plasmid DNA was isolated from these cells and the size of the inserted fragment was determined to be smaller than that of pMON5887 by restriction analysis using NcoI and NsiI. The nucleotide sequence of the region between NcoI and ClaI was determined and found to be that of the synthetic oligonucleotides. The new plasmid was designated pMON5967 and cells containing it were induced for protein production. Sonicated cell pellets and supernatants were used for protein purification and bio-assay.

EXAMPLE 6

Construction of pMON5978 which Encodes [Met-Ala-(15-125) hIL-3]

Plasmid DNA of pMON5967 isolated from

E. coli

GM48(dam-) was cleaved with ClaI and NsiI and ligated to 1 picomole of an annealed assembly of six oligonucleotides encoding hIL-3 amino acids 20-70 (FIG.

2

). This synthetic fragment encodes three unique restriction sites, EcoRV, XhoI and PstI. The sequence of these oligonucleotides is shown in FIG.

2

.

The resulting ligation mix was used to transform competent

E. coli

JM101 cells to ampicillin resistant colonies. Plasmid DNA was isolated and screened with XbaI and EcoRV for the presence of the new restriction site EcoRV. The DNA sequence of the region between ClaI and NsiI was determined and found to be the same as that of the synthetic oligonucleotides. The new plasmid was designated pMON5978, and cells containing it were induced for protein production. Sonicated cell pellets and supernatants were used for protein purification and bioassay.

Plasmid pMON5978 encodes [Met-Ala-(15-125) hIL-3] which has the following amino acid sequence:

[SEQ ID NO:13]

Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile

Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp

Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu

Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala

Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser

Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys

Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro

Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg

Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn

Ala Gln Ala Gln Gln

EXAMPLE 7

Construction of pMON5898

Plasmid pMON5851 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 3695 base pair NcoI,HindIII fragment. The genetic elements derived from pMON5851 are the beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, AraBAD promoter, g10L ribosome binding site and the lamB secretion leader. The AraBAD promoter is identical to that described in plasmid pMON6235 and the lamB signal peptide sequence used is that shown in

FIG. 8

fused to hIL-3 at the NcoI recognition site. Plasmid pMON5873 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 408 base pair NcoI,HindIII fragment. The genetic element derived from pMON5873 is the hIL-3 gene (1-133). Clones containing the hIL-3 (1-133) gene contained a 408 base pair NcoI, HindIII restriction fragment. This construct was designated pMON5898.

EXAMPLE 8

Construction of pMON5987

Plasmid pMON6458 DNA was digested with restriction enzymes NcoI and HindIII, resulting in a 3940 base pair NcoI, HindIII fragment. The genetic elements derived from pMON6458 are the beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, AraBAD promoter, g10L ribosome binding site and lamB secretion leader. Plasmid pMON5978 DNA was digested with NcoI and NsiI. The resulting 170 base pair NcoI, NsiI fragment encodes amino acids 15-71 of (15-125) hIL-3. Plasmid pMON5976 DNA was digested with NsiI and HindIII. The resulting 175 base pair NsiI, HindIII fragment encodes amino acids 72-125 of (15-125) hIL-3. The restriction fragments were ligated, and the ligation reaction mixture was used to transform

E. coli

K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and screened for the restriction sites EcoRV and NheI and DNA sequenced to confirm the correct insert.

EXAMPLE 9

Construction of pMON5988

The plasmid DNA of pMON5987 was digested with NheI and EcoRI, resulting in a 3903 base pair NheI, EcoRI fragment. The 3903 base pair NheI, EcoRI fragment was ligated to 1.0 picomoles of the following annealed oligonucleotides (oligo #3 and Oligo #4):

5′-CTAGCCACGGCCGCACCCACGCGACATCCAATCCATATCAA-3′-GGTGCCGGCGTGGGTGCGCTGTAGGTTAGGTATAGTTGGACGGTGACTGGAATG-3′ [SEQ ID NO:131]

CCTGCCACTGACCTTACAATT-5′ [SEQ ID NO:132]

The ligation reaction mixture was used to transform

E. coli

K-12 strain JM101 and transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm positive clones.

This plasmid was constructed to change alanine 101 to aspartic acid in the hIL-3 gene (15-125). The Ala

101

to Asp

101

change was confirmed by DNA sequencing. This plasmid was designated pMON5988 and encodes Peptide #1 [SEQ ID NO:65].

EXAMPLE 10

Construction of pMON5873 which encodes [Met-(1-133) hIL-3]

The gene obtained from British Biotechnology, Ltd. specified arginine at codon position 129. The amino acid specified in the native hIL-3 cDNA is serine. To produce a protein with the native sequence at this position, the portion of the coding sequence between the EcoRI site at codons 106 and 107 and the NheI site at codons 129 and 130 was replaced. Plasmid DNA of pMON5854 (Example 3) and pMON5853 (Example 64) were treated with EcoRI and NheI. The larger fragments of each were gel purified. These were ligated to a pair of an annealed oligonucleotides with the following sequences:

5′-AATTCCGTCGTAAACTGACCTTCTATCTGAAAACC-3′-GGCAGCATTTGACTGGAAGATAGACTTTTGGTTGGAGAACGCGCAGGCTCAACAGACCACTCTGTCG-3′ [SEQ ID NO: 136]

AACCTCTTGCGCGTCCGAGTTGTCTGGTGAGACAGCGATC-5′ [SEQ ID NO:137]

The ligation reaction mixtures were used to transform competent JM101 cells to ampicillin resistance. Colonies were picked into broth and grown. Plasmid DNA was isolated and screened for the presence of a new StyI recognition site present in the synthetic DNA and not in pMON5854 and pMON5853. The nucleotide sequence of the gene in the region between EcoRI and NheI was determined and found to be that of the synthetic oligonucleotides. The new plasmids were designated pMON5873 encoding [Met-(1-133) hIL-3] and pMON5872 encoding [Met-(15-133) hIL-3].

The plasmid, pMON5873, encodes Met-(1-133) hIL-3 which has the following amino acid sequence:

[SEQ ID NO:128]

Met Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr

Ser Trp Val Asn Cys Ser Asn Met Ile Asp Glu Ile

Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu

Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile

Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu

Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala

Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro

Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His

Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe

Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu

Asn Ala Gln Ala Gln Gln Thr Thr Leu Ser Leu Ala

Ile Phe

EXAMPLE 11

Construction of pMON6458

Plasmid pMON6525 DNA was digested with restriction enzymes HindIII and SalI and the resulting 3172 base pair fragment was isolated from a 1% agarose gel by interception onto DEAE membrane. The genetic elements derived from pMON6525 are the beta-lactamase gene (AMP), pBR327 origin of replication, and phage f1 origin of replication as the transcription terminator. (The genetic elements derived from plasmid pMON6525 are identical to those in plasmid pMON2341 which could also be used to construct pMON6458.) Plasmid pMON6457 was, digested with restriction enzymes HindIII and SalI and the resulting 1117 base pair fragment was isolated by PAGE and crush and soak elution. The genetic elements derived from pMON6457 are the pAraBAD promoter, g10L ribosome binding site, lamB secretion leader and the (15-125) hIL-3 gene. The restriction fragments were ligated and the ligation reaction mixture was used to transform

E. coli

K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes NcoI and HindIII in double digest. Clones containing the hIL-3 gene (encoding amino acids 15-125) contained a 345 base pair NcoI, HindIII restriction fragment. This construct was designated pMON6458. This plasmid was constructed to eliminate an EcoRI restriction site outside the hIL-3 gene coding region in plasmid pMON6457.

EXAMPLE 12

Construction of pMON6455

Plasmid pMON5905 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 3936 base pair fragment. The genetic elements derived from pMON5905 are the beta-lactamase gene (AMP), pBR327 origin of replication, pAraBAD promoter, g10L ribosome binding site, lamB secretion leader and phage f1 origin of replication as the transcription terminator. The following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, g10L ribosome binding site and phage f1 origin of replication as the transcription terminator, derived from plasmid pMON5905 are identical to those in plasmid pMON5594 which could also be used to construct pMON6455. The AraBAD promoter is identical to that described in pMON6235. The lamB signal peptide sequence used in pMON6455 is that shown in

FIG. 8

fused to hIL-3 (15-125) at the NcoI site. Plasmid pMON5887 DNA was digested with restriction enzymes HindIII and NcoI, resulting in a 384 base pair NcoI, HindIII fragment. The restriction fragments were ligated, and the ligation reaction mixture was used to transform into

E. coli

K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes NcoI and HindIII in double digest. Positive clones containing the hIL-3 gene (encoding amino acids 1-125) contained a 384 base pair NcoI, HindIII restriction fragment. This construct was designated pMON6455.

EXAMPLE 13

Construction of pMON6456

Plasmid pMON5905 DNA was digested with restriction enzymes HindIII and NcoI resulting in a 3936 base pair fragment. The genetic elements derived from pMONS905 are the beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, pAraBAD promoter, g10L ribosome binding site and the lamB secretion leader. Plasmid pMON5871 was digested with restriction enzymes HindIII and NcoI, resulting in a 330base pair NcoI, HindIII fragment. The genetic element derived from pMON5871 encompassed the bases encoding the (1-107) hIL-3 gene. The restriction fragments were ligated, and the ligation reaction mixture was used to transform

E. coli

K-12 strain JM101. Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes NcoI and HindIII in double digest. Clones containing the hIL-3 gene (encoding amino acids 1-107) contained a 330 base pair NcoI, HindIII restriction fragment. This construct was designated pMON6456.

EXAMPLE 14

Construction of pMON6457

Plasmid pMON6455 DNA grown in

E. coli

strain GM48 (dam-)was digested with restriction enzymes NcoI and ClaI, resulting in a 4293 base pair NcoI, ClaI fragment. The restriction fragment was ligated to 1.0 picomoles of annealed oligonucleotides (Oligo #5 and Oligo #6) with the following sequence coding for Met Ala 14-20 hIL-3:

5′-CATGGCTAACTGCTCTAACATGAT-3′[SEQ ID NO:151]

3′-CGATTGACCAGATTGTACTAGC-5′[SEQ ID NO:152]

The resulting DNA was transformed into

E. coli

K-12 strain JM101 and transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and the size of the inserted fragment was determined by restriction analysis employing restriction enzymes XbaI and EcoRI in double digest. Positive clones containing the hIL-3 gene (encoding aa 15-125 of hIL-3) contained a 433 base pair XbaI, EcoRI restriction fragment and were DNA sequenced. This construct was designated pMON6457. This plasmid was constructed to delete the first 14 amino acids of hIL-3. The coding sequence of the resulting gene begins as follows:

5′

ATG GCT AAC TGC . . .

3′

[SEQ ID NO:153]

Met Ala Asn Cys . . .

[SEQ ID NO:154]

15

The first two amino acids (Methionine, Alanine) create an NcoI restriction site and a signal peptidase cleavage site between the lamB signal peptide and (15-125) hIL-3. Plasmid pMON6457 encodes (15-125) hIL-3 which has the amino acid sequence designated SEQ ID NO 65.

EXAMPLE 15

Construction of pMON6235

One of the DNA fragments used to create this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the following oligonucleotides, Oligo #51(A) [SEQ ID NO:155] and Oligo #52(A) [SEQ ID NO:156], were used as primers in this procedure. The template for the PCR reaction was

E. coli

strain W3110 chromosomal DNA, prepared as described in Maniatis (1982). The oligonucleotide primers were designed to amplify the AraBAD promoter (Greenfield et al., 1978). The resulting DNA product was digested with the restriction enzymes SacII and BglII. The reaction mixture was purified as described previously. Plasmid, pMON5594, DNA was digested with SacII and BglII, resulting in a 4416 base pair SacII,BglII restriction fragment which contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, G10L ribosome binding site, phage f1 origin of replication as the transcription terminator and the chloramphenicol acetyl transferase (cat) gene. The 4416 base pair SacII,BglII restriction fragment from pMON5594 was ligated to the PCR-generated SacII, BglII DNA fragment. The ligation mixture was used to transform

E. coli

K-12 strain JM101. Positive clones contained a 323 base pair SacII,BglII fragment and were DNA sequenced to confirm that the SacII,BglII fragment was the AraBAD promoter. This construct was designated pMON6235.

EXAMPLE 16

Construction of pMON6460

One of the DNA fragments to construct this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the oligonucleotides, Oligo #7 [SEQ ID NO: 26] and Oligo #8 [SEQ ID NO: 27] as primers. The template for the PCR reaction was plasmid pMON6458 DNA. The resulting DNA product was digested with the restriction enzymes NcoI and EcoRI. Upon completion, the digest was heated at 70° C. for 15 minutes to inactivate the enzymes. The restriction fragment was purified by phenol/chloroform extraction and precipitation with equal volume isopropanol in the presence of 2M NH

4

OAc. The oligonucleotide, Oligo #8, introduces two stop codons (TAA) after amino acid 93 of hIL-3 and Creates a SalI restriction endonuclease recognition sequence. The NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones containing the above mentioned changes released a 1023 base pair SalI fragment. This construct was designated pMON6460. This plasmid was constructed to serve as the template for the creation of single amino acid substitution variants at positions 94, 95, 96 and 97 of hIL-3.

EXAMPLE 17

Construction of pMON6461

One of the DNA fragments to create this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the following oligonucleotide, Oligo #7 [SEQ. ID NO: 26] and Oligo #9 [SEQ. ID NO: 28], as primers. The template for the PCR reaction was plasmid pMON6458 DNA. The resulting DNA product was digested with the restriction enzymes NcoI and EcoRI. The oligonucleotide, Oligo #9, introduces two stop codons (TAA) after amino acid 97 of hIL-3 and creates a SalI restriction endonuclease recognition sequence. The NcoI, EcoRI restriction fragment from pMON5458 was ligated to the PCR-generated NcoI, EcoRI DNA fragment. Positive clones containing the above mentioned changes released a 1035 base pair SalI fragment. This construct was designated pMON6461. This plasmid was constructed to serve as the template for the creation of single amino acid substitution variants at positions 98, 99, 100 and 101 of hIL-3.

EXAMPLE 18

Construction of pMON6462

One of the DNA fragments to create this plasmid was generated by site-directed mutagenesis employing PCR techniques described previously using the following oligonucleotide, Oligo #7 [SEQ. ID NO: 26] and Oligo #10 [SEQ. ID NO: 31], as primers. The template for the PCR reaction was plasmid pMON6458 DNA. The resulting DNA product was digested with the restriction enzymes NcoI and EcoRI. The oligonucleotide, Oligo #10 [SEQ. ID NO: 31] introduces two stop codons (TAA) after amino acid 101 of hIL-3 and creates a SalI restriction endonuclease recognition sequence. The NcoI, EcoRI restriction fragment from pMON5458 was ligated to the PCR-generated NcoI, EcoRI DNA fragment. Positive clones containing the above mentioned changes released a 1047 base pair SalI fragment. This construct was designated pMON6462. This plasmid was constructed to serve as the template for the creation of single amino acid substitution variants at positions 102, 103, 104 and 105 of hIL-3.

EXAMPLE 19

Construction of Single Amino Acid Substitution Libraries at Positions 94, 95, 96 and 97

One of the DNA fragments used to construct the plasmids containing single amino acid substitution at positions 94, 95, 96 and 97 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction plasmid pMON6460 DNA was the template and the oligonucleotide, Oligo #7 [SEQ. ID NO: 26], was used as the primer at the N-terminus, The degenerate oligonucleotides, Oligo #11 [SEQ. ID NO: 32], Oligo #12 [SEQ. ID NO: 33], Oligo #13 [SEQ. ID NO: 34] and Oligo #14 [SEQ. ID NO: 35], were the primers at the C-terminus. These oligonucleotides are 32-fold degenerate, with G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 94, 95, 96 and 97 of hIL-3 respectively. These degenerate oligonucleotide primers theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at a single position. The degenerate oligonucleotides (Oligo #11 [SEQ. ID NO: 32], Oligo #12 [SEQ. ID NO: 33], Oligo #13 [SEQ. ID NO: 34] and Oligo #14 [SEQ. ID NO: 35]) replace the twelve bases introduced into pMON6460, that encode the two stop codons (TAA) after amino acid 93 of hIL-3 and the SalI recognition sequence. At the other 9 bases the DNA sequence was restored to encode the native hIL-3 protein sequence. The resulting PCR-generated DNA products were digested with the restriction enzymes NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6460 was ligated to the PCR-generated NcoI, EcoRI DNA fragments. Plasmid DNA from individual colonies was isolated as described previously and screened by DNA dot blot differential hybridization using the oligonucleotide, Oligo #15 [SEQ. ID NO: 36], as the probe which had been labeled with P

32

. Clones shown to be positive by hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 20

Construction of Single Amino Acid Substitution Libraries at Positions 98. 99, 100 and 101

Single amino acid substitutions variants were constructed at position 98, 99, 100 and 101 as described previously, with the following changes. In the PCR reaction the template was plasmid pMON6461 DNA and the oligonucleotide, Oligo #7 [SEQ. ID NO: 26], was used as the primer at the N-terminus. The degenerate oligonucleotides, Oligo #16 [SEQ. ID NO: 37], Oligo #17 [SEQ. ID NO: 38], Oligo #18 [SEQ. ID NO: 39] and Oligo #19 [SEQ. ID NO: 40], were used as primers at the C-terminus. The resulting PCR-generated DNA products were purified and digested with restriction enzymes NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6461 was ligated to the PCR-generated DNA NcoI, EcoRI restriction fragment. Single colonies were screened by DNA dot blot differential hybridization using the oligonucleotide, Oligo #20 [SEQ. ID NO: 41], as the probe. Clones shown to be positive by hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 21

Construction of Single Amino Acid Substitution Libraries at Positions 102, 103, 104 and 105

Single amino acid substitutions variants were constructed at position 102, 103, 104 and 105 as described previously, with the following changes. The template was pMON6462 and the oligonucleotide, Oligo #7 [SEQ. ID NO: 26], was used as the primer at the N-terminus. The degenerate oligonucleotides, Oligo #21 [SEQ. ID NO: 42], Oligo #22 [SEQ. ID NO: 43], Oligo #23 [SEQ. ID NO: 44] and Oligo #24 [SEQ. ID NO: 45] were used as primers at the C-terminus. The resulting PCR-generated DNA products were purified and digested with restriction enzymes, NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6462 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Single colonies were screened by DNA dot blot differential hybridization using the oligonucleotide, Oligo #25 [SEQ. ID NO: 46], as the probe. Clones shown to be positive by hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 22

Construction of Plasmid pMON6464

Amino acids 17-22 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the PCR reaction using the oligonucleotides, Oligo #26 and Oligo #27 as primers. The resulting PCR-generated DNA products were purified and digested with NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the bases encoding amino acids 17-22 of hIL-3 have been deleted. pMON6464 was made to serve as the template for the creation of single amino acid substitution variants at positions 17, 18, 19, 20, 21 and 22 of hIL-3.

EXAMPLE 23

Construction of Plasmid pMON6465

Amino acids 23-28 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo # 26 and Oligo #28, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the bases encoding amino acids 23-28 of hIL-3 have been deleted. pMON6465 was made to serve as the template for the creation of single amino acid substitution variants at positions 23, 24, 25, 26, 27 and 28 of hIL-3.

EXAMPLE 24

Construction of Plasmid pMON6466

Amino acids 29-34 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #29 as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRI. The 4008 bp NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the bases encoding amino acids 29-34 of hIL-3 have been deleted. pMON6466 was made to serve as the template for the creation of single amino acid substitution variants at positions 29, 30, 31, 32, 33 and 34 of hIL-3.

EXAMPLE 25

Construction of Plasmid pMON6467

Amino acids 35-40 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #30, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRV. The NcoI, EcoRV restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, EcoRV restriction fragment. Positive clones contained a 81 base pair NcoI, EcoRV restriction fragment in which the bases encoding amino acids 35-40 of hIL-3 have been deleted. pMON6467 was made to serve as the template for the creation of single amino acid substitution variants at positions 35, 36, 37, 38, 39 and 40 of hIL-3.

EXAMPLE 26

Construction of Plasmid pMON6468

Amino acids 41-46 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #31, as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and XhoI. The NcoI, XhoI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, XhoI restriction fragment. Positive clones contained a 119 base pair NcoI, XhoI restriction fragment in which the bases encoding amino acids 41-46 of hIL-3 have been deleted. pMON6468 was made to serve as the template for the creation of single amino acid substitution variants at positions 41, 42, 43, 44, 45 and 46 of hIL-3.

EXAMPLE 27

Construction of Plasmid pMON6469

Amino acids 47-52 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #32, as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and XhoI. The NcoI, XhoI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, XhoI restriction fragment. Positive clones contained a 119 base pair NcoI, XhoI restriction fragment in which the bases encoding amino acids 47-52 of hIL-3 have been deleted. pMON6469 was made to serve as the template for the creation of single amino acid substitution variants at positions 47, 48, 49, 50, 51 and 52 of hIL-3.

EXAMPLE 28

Construction of Plasmid pMON6470

Amino acids 53-58 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid, pMON5988, DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #33, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and NsiI. The NcoI, NsiI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, NsiI restriction fragment. Positive clones contained a 152 base pair NcoI, NsiI restriction fragment in which the bases encoding amino acids 53-58 of hIL-3 have been deleted. pMON6470 was made to serve as the template for the creation of single amino acid substitution variants at positions 53, 54, 55, 56, 57 and 58 of hIL-3.

EXAMPLE 29

Construction of Plasmid pMON6471

Amino acids 59-64 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #34, as the primers. The resulting PCR-generated DNA product was purified and digested with NcoI and NsiI. The NcoI, NsiI restriction fragment from pMON5988 was ligated to the PCR-generated NcoI, NsiI restriction fragment. Positive clones contained a 152 base pair NcoI, NsiI restriction fragment in which the bases encoding amino acids 59-64 of hIL-3 have been deleted. pMON6471 was made to serve as the template for the creation of single amino acid substitution variants at positions 59, 60, 61, 62, 63 and 64 of hIL-3.

EXAMPLE 30

Construction of Plasmid pMON6472

Amino acids 65-70 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #35, as primers. The resulting PCR-generated DNA product was purified and digested with EcoRI and XhoI. The EcoRI, XhoI restriction fragment from pMON5988 was ligated to the PCR-generated EcoRI, XhoI restriction fragment. Positive clones contained a 126 base pair EcoRI, XhoI restriction fragment in which the bases encoding amino acids 65-70 of hIL-2 have been deleted. pMON6472 was made to serve as the template for the creation of single amino acid substitution variants at positions 65, 66, 67, 68, 69 and 70 of hIL-3.

EXAMPLE 31

Construction of Plasmid pMON6473

Amino acids 71-76 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid, pMON5988, DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #36, as primers. The resulting PCR-generated DNA product was and digested with PstI and EcoRI. The PstI, EcoRI restriction fragment from pMON5988 was ligated to the PCR-generated PstI, EcoRI restriction fragment. Restriction analysis was with NcoI, NsiI and EcoRI in a triple digest. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment, in which the bases encoding amino acids 71-76 of hIL-3 have been deleted, and lost the NsiI restriction site. pMON6473 was made to serve as the template for the creation of single amino acid substitution variants at positions 71, 72, 73, 74, 75 and 76 of hIL-3.

EXAMPLE 32

Construction of Plasmid pMON6474

Amino acids 77-82 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #37, as primers. The resulting PCR-generated DNA product was purified and digested with PstI and EcoRI. The PstI, EcoRI restriction fragment from pMON5988 was ligated to the PCR-generated PstI, EcoRI restriction fragment. Restriction analysis was with NcoI, NsiI and EcoRI in a triple digest. Positive clones contained a 170 base pair NcoI, NsiI restriction fragment and a 93 base pair NsiI, EcoRI restriction fragment in which the bases encoding amino acids 77-82 of hIL-3 have been deleted. pMON6474 was made to serve as the template for the creation of single amino acid substitution variants at positions 77, 78, 79, 80, 81 and 82 of hIL-3.

EXAMPLE 33

Construction of Plasmid pMON6475

Amino acids 83-88 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON5988 DNA was the template in the reaction using the oligonucleotides, Oligo #26 and Oligo #38, as primers. The resulting PCR-generated DNA product was digested with PstI and EcoRI. The PstI, EcoRI restriction fragment from pMON5988 was ligated to the PCR-generated PstI, EcoRI restriction fragment. Restriction analysis was with NcoI, NsiI and EcoRI in a triple digest. Positive clones contained a 170 base pair NcoI, NsiI restriction fragment and a 93 base pair NsiI, EcoRI restriction fragment in which the bases encoding amino acids 83-88 of hIL-3 have been deleted. pMON6475 was made to serve as the template for the creation of single amino acid substitution variants at positions 83, 84, 85, 86, 87 and 88 of hIL-3.

EXAMPLE 34

Construction of Plasmid pMON6476

Amino acids 88-93 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #39, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and EcoRI. The NcoI, EcoRI restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, EcoRI restriction fragment. Positive clones contained a 263 base pair NcoI, EcoRI restriction fragment in which the-bases encoding amino acids 88-93 of hIL-3 have been deleted. pMON6476 was made to serve as the template for the creation of single amino acid substitution variants at positions 88, 89, 90, 91, 92 and 93 of hIL-3.

EXAMPLE 35

Construction of Plasmid pMON6477

Amino acids 106-111 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #40, as primers. The resulting PCR-generated DNA fragment was purified and digested with NcoI and HindIII. The NcoI, HindIII restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, HindIII restriction fragment. Positive clones contained a 327 base pair NcoI, HindIII restriction fragment in which the bases encoding amino acids 106-111 of hIL-3 have been deleted. pMON6477 was made to serve as the template for the creation of single amino acid substitution variants at positions 106, 107, 108, 109, 110 and 111 of hIL-3.

EXAMPLE 36

Construction of Plasmid pMON6478

Amino acids 112-117 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, Oligo #7 and Oligo #41, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and HindIII. The 4008 bp NcoI, HindIII restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, HindIII restriction fragment. Positive clones contained a 327 base pair NcoI, HindIII restriction fragment in which the bases encoding amino acids 112-117 of hIL-3 have been deleted. pMON6478 was made to serve as the template for the creation of single amino acid substitution variants at positions 112, 113, 114, 115, 116 and 117 of hIL-3.

EXAMPLE 37

Construction of Plasmid pMON6479

Amino acids 118-123 of hIL-3 were deleted using site-directed PCR mutagenesis methods described previously. Plasmid pMON6458 DNA was the template in the reaction using the oligonucleotides, oligo #7 and Oligo #42, as primers. The resulting PCR-generated DNA product was purified and digested with NcoI and HindIII. The NcoI, HindIII restriction fragment from pMON6458 was ligated to the PCR-generated NcoI, HindIII restriction fragment. Positive clones contained a 327 base pair NcoI, HindIII restriction fragment in which the bases encoding amino acids 118-123 of hIL-3 have been deleted. pMON6479 was made to serve as the template for the creation of single amino acid substitution variants at positions 118, 119, 120, 121, 122 and 123 of hIL-3.

EXAMPLE 38

Construction of Single Amino Acid Substitution Libraries at Positions 17, 18, 19, 20, 21 and 22

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 17, 18, 19, 20, 21 and 22 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6464 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #43, Oligo #44, Oligo #45, Oligo #46, Oligo #47 and Oligo #48 were the primers at the C-terminus. The oligonucleotide, Oligo #26, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6464. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 17, 18, 19, 20, 21 and 22 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA product was digested with NcoI and EcoRV. Plasmid pMON6464 DNA was digested with restriction enzymes NcoI and EcoRV resulting in a 4190 base pair fragment which was ligated to the PCR-generated NcoI, EcoRV restriction fragments. Plasmid DNA was isolated and screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #139, which had been labeled with P

32

. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 39

Construction of Single Amino Acid Substitution Libraries at Positions 23, 24, 25, 26, 27 and 28

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 23, 24, 25, 26, 27 and 28 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6465 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #49, Oligo #50, Oligo #51, Oligo #52, Oligo #53 and Oligo #54 were the primers at the C-terminus. The oligonucleotide, Oligo #26, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6465. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 23, 24, 25, 26, 27 and 28 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with restriction enzymes NcoI and EcoRV. Plasmid pMON6465 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 4190 base pair fragment was ligated to the PCR-generated NcoI, EcoRV DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #140, which had been labeled with P

32

. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 40

Construction of Single Amino Acid Substitution Libraries at Positions 29, 30, 31, 32, 33 and 34

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 29, 30, 31, 32, 33 and 34 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6466 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #55, Oligo #56, Oligo #57, Oligo #58, Oligo #59 and Oligo #60 were the primers at the C-terminus. The oligonucleotide Oligo #26 was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6466. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 29, 30, 31, 32, 33 and 34 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and EcoRV. Plasmid pMON6466 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 4190 base pair fragment was ligated to the PCR-generated NcoI, EcoRV DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #141, which had been labeled with P

32

. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 41

Construction of Single Amino Acid Substitution Libraries at Positions 35, 36, 37, 38, 39 and 40

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 35, 36, 37, 38, 39 and 40 of hIL-3 were generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6467 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #61, Oligo #62, Oligo #63, Oligo #64, Oligo #65 and Oligo #66 were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6467. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 35, 36, 37, 38, 39 and 40 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored and at the other position, 32 different codons substitutions were created at positions independently. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and EcoRV. Plasmid pMON6467 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 4190 base pair fragment was ligated to the PCR-generated NcoI, EcoRV DNA fragments.

Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #142, which had been labeled with p32. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 42

Construction of Single Amino Acid Substitution Libraries at Positions 41, 42, 43, 44, 45 and 46

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 41, 42, 43, 44, 45 and 46 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6468 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #67, Oligo #68, Oligo #69, Oligo #70, Oligo #71 and Oligo #72 were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6468. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 41, 42, 43, 44, 45 and 46 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and XhoI. Plasmid pMON6468 DNA was digested with restriction enzymes NcoI and XhoI and the resulting 4152 base pair fragment was ligated to the PCR-generated NcoI, XhoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #143, which had been labeled with P

32

. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 43

Construction of Single Amino Acid Substitution Libraries at Positions 47, 48, 49, 50, 51 and 52

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 47, 48, 49, 50, 51 and 52 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6469 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #73, Oligo #74, Oligo #75, Oligo #76, Oligo #77 and Oligo #78 , were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6469. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 47, 48, 49, 50, 51 and 52 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and XhoI. Plasmid pMON6469 DNA was digested with restriction enzymes NcoI and XhoI and the resulting 4152 base pair fragment was ligated to the PCR-generated NcoI, XhoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #143, which had been labeled with P

32

. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 44

Construction of Single Amino Acid Substitution Libraries at Positions 53, 54, 55, 56, 57 and 58

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 53, 54, 55, 56, 57 and 58 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6470 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #79, Oligo #80, Oligo #81, Oligo #82, Oligo #83 and Oligo #84 , were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6470. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 53, 54, 55, 56, 57 and 58 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and NsiI. Plasmid pMON6470 DNA was digested with restriction enzymes NcoI and NsiI and the resulting 4119 base pair fragment was ligated to the PCR-generated NcoI, NsiI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #145, which had been labeled with P

32

. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 45

Construction of Single Amino Acid Substitution Libraries at Positions 59, 60, 61, 62, 63 and 64

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 59, 60, 61, 62, 63 and 64 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6471 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #85, Oligo #86, Oligo #87, Oligo #88, Oligo #89 and Oligo #90 , were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6471. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 59, 60, 61, 62, 63 and 64 of hIL-3 respectively, These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes NcoI and NsiI. Plasmid pMON6471 DNA was digested with restriction enzymes NcoI and NsiI and the resulting 4119 base pair fragment was ligated to the PCR-generated NcoI, NsiI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #146, which had been labeled with P

32

. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 46

Construction of Single Amino Acid Substitution Libraries at Positions 65, 66, 67, 68, 69 and 70

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 65, 66, 67, 68, 69 and 70 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6472 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #91, Oligo #92, Oligo #93, Oligo #94, Oligo #95 and Oligo #96 , were the primers at the N-terminus. The oligonucleotide, Oligo 26, was used as the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6472. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 65, 66, 67, 68, 69 and 70 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes EcoRI and XhoI. Plasmid pMON6472 DNA was digested with restriction enzymes EcoRI and XhoI and the resulting 4145 base pair fragment was ligated to the PCR-generated EcoRI, XhoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #147, which had been labeled with P

32

. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 47

Construction of Single Amino Acid Substitution Libraries at Positions 71, 72, 73, 74, 75 and 76

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 71, 72, 73, 74, 75 and 76 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6473 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #97, Oligo #98, Oligo #99, Oligo #100, Oligo #101 and Oligo #102 , were the primers at the N-terminus. The oligonucleotide, Oligo #26, was used as the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6473. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 71, 72, 73, 74, 75 and 76 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA fragments were purified and digested with the restriction enzymes EcoRI and PstI. Plasmid pMON6473 DNA was digested with restriction enzymes EcoRI and PstI and the resulting 4171 base pair fragment was ligated to the PCR-generated EcoRI, PstI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #148, which had been labeled with P

32

. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 48

Construction of Single Amino Acid Substitution Libraries at Positions 77, 78, 79, 80, 81 and 82

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 77, 78, 79, 80, 81 and 82 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the reaction the plasmid pMON6474 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #103, Oligo #104, Oligo #105, Oligo #106, Oligo #107 and Oligo #108, were the primers at the N-terminus. The oligonucleotide, Oligo #26, was used as the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6474. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 77, 78, 79, 80, 81 and 82 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes EcoRI and PstI as described previosly. Plasmid pMON6474 DNA was digested with restriction enzymes EcoRI and PstI and the resulting 4171 base pair fragment was ligated to the PCR-generated EcoRI, PstI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #149, which had been labeled with P

32

. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 49

Construction of Single Amino Acid Substitution Libraries at Positions 83, 84, 85, 86, 87 and 88

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 83, 84, 85, 86, 87 and 88 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6475 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #109, Oligo #110, Oligo #111, Oligo #112, Oligo #113 and Oligo #114, were the primers at the N-terminus. The oligonucleotide, Oligo #26, was used an the primer at the C-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6475. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 83, 84, 85, 86, 87 and 88 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA producs were purified and digested with the restriction enzymes EcoRI and PstI. Plasmid pMON6475 DNA was digested with restriction enzymes EcoRI and PstI and the resulting 4171 base pair fragment was ligated to the PCR-generated EcoRI, PstI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #150, which had been labeled with P

32

. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 50

Construction of Single Amino Acid Substitution Libraries at Positions 88, 89, 90, 91, 92 and 93

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 88, 89, 90, 91, 92 and 93 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6476 DNA was the template and the following degenerate oligonucleotides, Oligo #114, Oligo #115, Oligo #116, Oligo #117, Oligo #118 and Oligo #119, were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6476. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 88, 89, 90, 91, 92 and 93 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes EcoRI and NcoI. Plasmid pMON6476 DNA was digested with restriction enzymes EcoRI and NcoI and the resulting 4008 base pair fragment was ligated to the PCR-generated EcoRI, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #151, which had been labeled with P

32

. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 51

Construction of Single Amino Acid Substitution Libraries at Positions 106, 107, 108, 109, 110 and 111

One of the DNA fragments used to construct the plasmids containing the single amino acid substitutions at positions 106, 107, 108, 109, 110 and 111 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously in two sequential PCR reactions. In the first PCR reaction, plasmid pMON6477 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #120, Oligo #121, Oligo #122, Oligo #123, Oligo #124 and Oligo #125 were the primers at the C-terminus. The oligonucleotide, Oligo #7 was the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON9477. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 106, 107, 108, 109, 110 and 111 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The DNA generated in this PCR reaction was purified by phenol/chloroform extraction and precipitation with equal volume isopropanol in the presence of 2M NH

4

OAc to remove any primer that was not extended. This DNA was then used as a primer in the second PCR reaction.

In the second PCR reaction plasmid pMON6477 DNA was the template, the DNA product generated in the first PCR reaction (described above) was the primer at the N-terminus and the oligonucleotide, Oligo #126 (DNA sequence shown in Table 1), was the primer at the C-terminus. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes HindIII and NcoI. Plasmid pMON6477 was digested with restriction enzymes HindIII and NcoI and the resulting 3944 base pair fragment was ligated to the PCR-generated HindIII, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #152, which had been labeled with P

32

. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 52

Construction of Single Amino Acid Substitution Libraries at Positions 112, 113, 114, 115, 116 and 117

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 112, 113, 114, 115, 116 and 117 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6478 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #127, Oligo #128, Oligo #129, Oligo #130, Oligo #131 and Oligo #132, were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus, The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6478. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 112, 113, 114, 115, 116 and 117 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes HindIII and NcoI. Plasmid pMON6478 was digested with restriction enzymes HindIII and NcoI and the resulting 3944 base pair fragment was ligated to the PCR-generated HindIII, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #153, which had been labeled with P

32

. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 53

Construction of Single Amino Acid Substitution Libraries at Positions 118, 119, 120, 121, 122 and 123

One of the DNA fragments used to construct the plasmids containing single amino acid substitutions at positions 118, 119, 120, 121, 122 and 123 of hIL-3 was generated by site-directed mutagenesis employing PCR techniques described previously. In the PCR reaction the plasmid pMON6479 DNA was the template and the following 32 fold degenerate oligonucleotides, Oligo #133, Oligo #134, Oligo #135, oligo #136, Oligo #137 and Oligo #138, were the primers at the C-terminus. The oligonucleotide, Oligo #7, was used as the primer at the N-terminus. The degenerate oligonucleotides replace the eighteen bases, encoding six amino acids, deleted in pMON6479. The degenerate oligonucleotides have G, A, T or C in the first and second positions and G or C in the third position of a single codon at amino acid positions 118, 119, 120, 121, 122 and 123 of hIL-3 respectively. These degenerate oligonucleotide primers result in libraries which theoretically contain 32 different codons encoding all 20 amino acid substitutions and one translational stop codon at one position. At the other five amino acid positions the native hIL-3 DNA sequence was restored. The resulting PCR-generated DNA products were purified and digested with the restriction enzymes HindIII and NcoI. Plasmid pMON6479 DNA was digested with restriction enzymes HindIII and NcoI and the resulting 3944 base pair fragment was ligated to the PCR-generated HindIII, NcoI DNA fragments. Transformant bacteria were screened by DNA dot blot differential hybridization using the oligonucleotide probe, Oligo #154, which had been labeled with P

32

. Clones shown to be positive by colony hybridization were selected, plasmid DNA isolated and DNA sequenced to determine the amino acid substitution.

EXAMPLE 54

Construction of pMON13358

Plasmid pMON5978 DNA (Example 6) was digested with restriction enzymes NsiI and EcoRI and the resulting 3853 base pair NsiI,EcoRI fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, recA promoter, g10L ribosome binding site and the bases encoding amino acids 15-71 and 106-125 of (15-125) hIL-3. The 3853 base pair NsiI,EcoRI restriction fragment from pMON5978 was ligated to the following annealed complementary oligonucleotides.

Oligo #15(A) [SEQ ID NO: 29]

Oligo #16(A) [SEQ ID NO: 30]

In the resulting plasmid the 111 bases between the NsiI and EcoRI restriction sites in the (15-125) hIL-3 gene are replaced with 24 bases from the above mentioned oligonucleotides. This linker also creates a NdeI recognition sequence.

EXAMPLE 55

Construction of pMON13304

Plasmid pMON13358 DNA is digested with restriction enzymes PstI and EcoRI and the resulting 3846 base pair PstI,EcoRI fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, recA promoter, g10L ribosome binding site and the bases encoding amino acids 15-69 and 106-125 of (15-125) hIL-3. The 3846 base pair NsiI,EcoRI restriction fragment from pMON13358 is ligated to the following annealed complementary oligonucleotides.

Oligo #155

[SEQ ID NO:200]

Oligo #156

[SEQ ID NO:201]

Oligo #157

[SEQ ID NO:202]

Oligo #158

[SEQ ID NO:203]

Oligo #159

[SEQ ID NO:204]

Oligo #160

[SEQ ID NO:205]

Oligo #161

[SEQ ID NO:206]

Oligo #162

[SEQ ID NO:207]

When assembled, the oligonucleotides create PstI and EcoRI restriction ends and the DNA sequence that encodes amino acids 70-105 of (15-125) hIL-3 with the following amino acid substitutions; 98I and 100R. The codons encoding amino acids 70-105 of (15-125) hIL-3 are those found in the hIL-3 cDNA sequence except at those positions where amino acid substitutions were made. The plasmid, pMON13304, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

Peptide #A1 [SEQ ID NO:66]

EXAMPLE 56

Construction of pMON13305

Plasmid pMON13358 DNA is digested with restriction enzymes PstI and EcoRI and the resulting 3846 base pair PstI, EcoRI fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, recA promoter, g10L ribosome binding site and the bases encoding amino acids 15-69 and 106-125 of (15-125) hIL-3. The 3846 base pair NsiI,EcoRI restriction fragment from pMON13358 is ligated to the following annealed complementary oligonucleotides.

Oligo #155

[SEQ ID NO:200]

Oligo #156

[SEQ ID NO:201]

Oligo #157

[SEQ ID NO:202]

Oligo #158

[SEQ ID NO:203]

Oligo #159

[SEQ ID NO:204]

Oligo #160

[SEQ ID NO:205]

Oligo #163

[SEQ ID NO:208]

Oligo #164

[SEQ ID NO:209]

When assembled, the oligonucleotides create PstI and EcoRI restriction ends and the DNA sequence that encodes amino acids 70-105 of (15-125) hIL-3 with the following amino acid substitutions; 95R, 98I and 100R. The codons encoding amino acids 70-105 of (15-125) hIL-3 are those found in the hIL-3 cDNA sequence except at those positions where amino acid substitutions were made. The plasmid, pMON13305, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

Peptide #A2 [SEQ ID NO:67]

EXAMPLE 57

Construction of pMON13286

Plasmid pMON5978 DNA was digested with restriction enzymes NcoI and EcoRV and the resulting 3865 base pair NcoI,EcoRV fragment contains the following genetic elements; beta-lactamase gene (AMP), pBR327 origin of replication, phage f1 origin of replication as the transcription terminator, precA promoter, g10L ribosome binding site and the bases encoding amino acids 47-125 of (1-125) hIL-3. The 3865 base pair NcoI,EcoRV restriction fragment from pMON5978 was ligated to the following annealed complementary oligonucleotides.

Oligo #165

[SEQ ID NO:210]

Oligo #166

[SEQ ID NO:211]

Oligo #167

[SEQ ID NO: 212]

Oligo #168

[SEQ ID NO:213]

Oligo #169

[SEQ ID NO:214]

Oligo #170

[SEQ ID NO:215]

When assembled, the oligonucleotides create NcoI and EcoRV restriction ends and the DNA sequence that encodes amino acids 15-46 of (15-125) hIL-3 with the following amino acid substitutions; 42D, 45M and 46S. The codons encoding amino acids 15-46 of (15-125) hIL-3 are those found in the hIL-3 cDNA sequence except at those positions where amino acid substitutions were made. The plasmid, pMON13286, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

Peptide #A3 SEQ ID NO:69

DNA sequence #A4 pMON13286 42D, 45M, 46S

ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC

ACCTGAAGCA GCCACCGCTG CCGCTGCTGG ACTTCAACAA

CCTCAATGAC GAAGACATGT CTATCCTGAT GGAAAATAAC

CTTCGTCGTC CAAACCTCGA GGCATTCAAC CGTGCTGTCA

AGTCTCTGCA GAATGCATCA GCAATTGAGA GCATTCTTAA

AAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC

ACGCGACATC CAATCCATAT CAAGGACGGT GACTGGAATG

AATTCCGTCG TAAACTGACC TTCTATCTGA AAACCTTGGA

GAACGCGCAG GCTCAACAG SEQ ID NO:68

EXAMPLE 58

Construction of pMON5853 (

FIG. 6

) which Encodes [Met-(15-133) hIL-3 (Arg129)]

Plasmid DNA of pMON5847 (Example 2) was treated with NcoI. The restriction enzyme was inactivated by heat treatment (65° C. for 10 minutes). The DNA was then treated with large fragment of DNA polymerase I (Klenow) in the presence of all four nucleotide precursors. This produces DNA termini with non-overlapping ends. After 5 minutes at 37° C., the polymerase was inactivated by heat treatment at 65° C. for 10 minutes. The DNA was then treated with HpaI, an enzyme which produces non-overlapping termini. The DNA was ethanol precipitated and ligated. The ligation reaction mixture was used to transform competent JM101 cells to ampicillin resistance. Colonies were picked and plasmid DNA was analyzed by restriction analysis. A plasmid designated pMON5853 was identified as one containing a deletion of the amino terminal 14 codons of the hIL-3 gene. The DNA sequence for the junction of the ribosome binding site to the (15-133) hIL-3 gene was determined to be the following:

5′-AAGGAGATATATCCATGAACTGCTCTAAC-3′

[SEQ ID NO:133]

M N C S N

[SEQ ID NO:134]

The lower line contains the one-letter code for the amino acids specified by the coding sequence of the amino terminus of the 15-133 hIL-3 gene. These are methionine, asparagine, cysteine, serine and asparagine.

When cultures of JM101 cells harboring this plasmid were induced with nalidixic acid, it was found that hIL-3 (15-133) accumulated at levels higher than hIL-3 (pMON5847).

The plasmid, pMON5853, encodes Met-(15-133) hIL-3 (Arg

129

) which has the following amino acid sequence:

Met Asn Cys Ser Asn Met Ile Asp Glu Ile Ile

Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu

Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile

Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu

Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala

Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro

Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His

Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe

Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu

Asn Ala Gln Ala Gln Gln Thr Thr Leu Arg Leu Ala

Ile Phe [SEQ ID NO:135]

Formula XI shown below is a representation of a [(15-125) hIL-3 mutein] with numbers in bold type added above the amino acids to represent the position at which the amino acid below the bolded number appears in native (1-133) hIL-3 [e.g. the amino acid at position 1 of Formula XI corresponds to the Asn which appears at position 15 in native (1-133) hIL-3]. The number shown in bold indicates the amino acids that correspond to the native IL-3(1-133). The non-bold members below the amino acids sequences are for Seq Id reference numbers. When the muteins are expressed the initial amino acid may be preceded by Met- or Met-Ala-.

15 20 25

Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln

[SEQ ID NO:23]

1 5 10 15

30 35 40

Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp

20 25 30

45 50 55

Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu

35 40 45

60 65 70

Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile

50 55 60

75 80 85

Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr

65 70 75

90 95 100

Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp

80 85 90

105 110 115

Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu

95 100 105

120 125

Asn Ala Gln Ala Gln Gln

110

Table 6 shows (15-125) hIL-3 muteins of the present invention which have one (and in some cases two) amino acid substitutions in the (15-125) hIL-3 polypeptide and which were constructed as described in the Examples. The mutants in Table 6 were secreted into the periplasmic space in

E. coli.

The periplasmic content was released by osmotic shock and the material in the crude osmotic shock fraction was screened for growth promoting activity. Biological activity is the growth promoting activity of AML cells relative to (15-125) hIL-3 (pMON6458 or pMMON5988). The numbers in parentheses indicate the number of repeat assays. When a variant was assayed more than once the standard deviation is indicated. An “-” indicates that the hIL3 variant protein level was less than 1.0 μg/ml and was not screened for growth promoting activity.

TABLE 6

(15-125) HUMAN INTERLEUKIN-3 MUTANTS

hIL-3 aa

PARENTAL

(15-125)hIL-3 MUTANT

POSITION

1

aa

CODON

aa

SEQ ID NO:

CODON

BIOL ACTIVITY

17/3

1

SER

TCT

LYS

19

AAG

<0.018 (1)

17/3

SER

TCT

GLY

19

GGG

1.2 ± 1.1 (3)

17/3

SER

TCT

ASP

19

GAC

1.0 ± 0.7 (3)

17/3

SER

TCT

MET

19

ATG

0.50 (1)

17/3

SER

TCT

GLN

19

CAG

1.2 ± 0.7 (3)

17/3

SER

TCT

ARG

19

AGG

<0.070 (1)

18/4

ASN

AAC

HIS

19

CAC

1.2 ± 0.3 (3)

18/4

ASN

AAC

LEU

19

CTC

0.45 ± 0.42 (4)

18/4

ASN

AAC

ILE

19

ATC

1.5 ± 0.2 (2)

18/4

ASN

AAC

PHE

19

TTC

0.19 ± 0.26 (2)

18/4

ASN

AAC

ARG

19

CGG

0.10 (1)

18/4

ASN

AAC

GLN

19

CAA

0.37 (1)

19/5

MET

ATG

PHE

19

TTC

0.25 (1)

19/5

MET

ATG

ILE

19

ATG

0.77 ± 0.70 (9)

19/5

MET

ATG

ARG

19

AGG

0.17 (1)

19/5

MET

ATG

GLY

19

GGA

0.06 (1)

19/5

MET

ATG

ALA

19

GCG

0.19 (1)

19/5

MET

ATG

CYS

19

TGC

—

20/6

ILE

ATC

CYS

19

TGC

—

20/6

ILE

ATC

GLN

19

CAG

—

20/6

ILE

ATC

GLU

19

GAG

<0.025 (1)

20/6

ILE

ATC

ARG

19

CGC

<0.025 (1)

20/6

ILE

ATC

PRO

19

CCG

0.29 ± 0.16 (3)

20/6

ILE

ATC

ALA

19

GCG

0.18 (1)

21/7

ASP

GAT

PHE

19

TTC

<0.016 (1)

21/7

ASP

GAT

LYS

19

AAG

0.027 ± 0.027 (2)

21/7

ASP

GAT

ARG

19

AGG

<0.008 (1)

21/7

ASP

GAT

ALA

19

GCG

0.07 ± 0.06 (3)

21/7

ASP

GAT

GLY

19

GGG

0.032 (1)

21/7

ASP

GAT

VAL

19

GTG

<0.008 (1)

22/8

GLU

GAA

TRP

19

TGG

—

22/8

GLU

GAA

PRO

19

CCG

<0.015 (1)

22/8

GLU

GAA

SER

19

TCG

<0.015 (1)

22/8

GLU

GAA

ALA

19

GCC

<0.015 (1)

22/8

GLU

GAA

HIS

19

CAC

<0.015 (1)

22/8

GLU

GAA

GLY

19

GGC

<0.008 (1)

23/9

ILE

ATT

VAL

19

GTG

0.18 (1)

23/9

ILE

ATT

ALA

2

19

GCG

1.16 ± 0.16 (3)

23/9

ILE

ATT

LEU

19

TTG

1.3 (1)

23/9

ILE

ATT

GLY

2

19

GGG

0.06 (1)

23/9

ILE

ATT

TRP

19

TGG

—

23/9

ILE

ATT

LYS

2

19

AAG

—

23/9

ILE

ATT

PHE

19

TTC

—

23/9

ILE

ATT

LEU

2

19

TTG

3.0 ± 1.1 (3)

23/9

ILE

ATT

SER

2

19

AGC

<0.005 (1)

23/9

ILE

ATT

ARG

2

19

CGC

—

24/10

ILE

ATA

GLY

19

GGG

<0.004 (1)

24/10

ILE

ATA

VAL

19

GTC

0.89 ± 0.23 (4)

24/10

ILE

ATA

ARG

3

19

CGG

—

24/10

ILE

ATA

SER

19

AGC

<0.003 (1)

24/10

ILE

ATA

PHE

19

TTC

0.29 ± 0.24 (2)

24/10

ILE

ATA

LEU

19

CTG

0.52 ± 0.12 (3)

25/11

THR

ACA

HIS

19

CAC

1.11 ± 0.2 (3)

25/11

THR

ACA

GLY

19

GGC

0.48 ± 0.27 (4)

25/11

THR

ACA

GLN

19

CAG

1.0 ± 0.8 (4)

25/11

THR

ACA

ARG

19

CGG

0.26 ± 0.17 (2)

25/11

THR

ACA

PRO

19

CCG

0.36 (1)

31/17

PRO

CCT

GLY

19

GGG

0.79 ± 0.61 (2)

31/17

PRO

CCT

ALA

19

GCC

0.49 (1)

31/17

PRO

CCT

ARG

19

CGC

0.25 ± 0.20 (2)

31/17

PRO

CCT

LEU

19

CTG

0.22 (1)

31/17

PRO

CCT

GLN

19

CAG

0.62 ± 0.04 (2)

31/17

PRO

CCT

LEU

4

19

CTG

0.30 ± 0.20 (3)

32/18

LEU

TTG

VAL

2

19

GTG

0.01 (1)

32/18

LEU

TTG

ARG

19

CCC

1.5 ± 1.0 (4)

32/18

LEU

TTG

CLN

19

CAG

0.93 ± 0.18 (3)

32/18

LEU

TTG

ASN

19

AAC

1.2 ± 0.5 (5)

32/18

LEU

TTG

GLY

5

19

GGC

0.84 ± 1.0 (3)

32/18

LEU

TTG

ALA

19

GCG

1.4 ± 0.7 (5)

32/18

LEU

TTG

GLU

19

GAG

0.88 ± 0.37 (2)

33/19

PRO

CC(T/C)

LEU

19

CTG

0.13 (1)

33/19

PRO

CC(T/C)

GLN

19

CAG

0.22 ± 0.20 (2)

33/19

PRO

CC(T/C)

ALA

19

GCG

0.30 ± 0.14 (2)

33/19

PRO

CC(T/C)

THR

19

ACC

<0.018 (1)

33/19

PRO

CC(T/C)

GLU

19

GAG

0.54 ± 0.43 (2)

34/20

LEU

TTG

VAL

19

GTG

1.2 ± 0.6 (3)

34/20

LEU

TTG

GLY

19

GGG

0.64 ± 0.74 (2)

34/20

LEU

TTG

SER

19

TCG

1.5 ± 0.7 (4)

34/20

LEU

TTG

LYS

19

AAG

0.97 ± 0.28 (2)

34/20

LEU

TTG

MET

19

ATG

1.1 ± 0.5 (3)

35/21

LEU

CTG

ALA

19

GCC

1.6 ± 0.5 (3)

35/21

LEU

CTG

GLY

19

GGC

<0.006 ± 0.002 (3)

35/21

LEU

CTG

ASN

19

AAC

1.1 ± 1.7 (5)

35/21

LEU

CTG

PRO

19

CCC

1.8 ± 2.0 (5)

35/21

LEU

CTG

GLN

19

CAA

0.98 ± 1.1 (5)

35/21

LEU

CTG

VAL

19

GTG

0.76 ± 0.86 (5)

36/22

ASP

GAC

LEU

19

CTC

0.20 (1)

25/11

THR

ACA

ALA

19

GCC

0.86 ± 0.27 (3)

26/12

HIS

CAC

THR

19

ACG

0.010 (1)

26/12

HIS

CAC

PHE

19

TTC

0.26 (1)

26/12

HIS

CAC

GLY

19

GGG

0.19 (1)

26/12

HIS

CAC

ARG

19

CGG

0.21 (1)

26/12

HIS

CAC

ALA

19

GCC

0.56 ± 0.03 (2)

26/12

HIS

CAC

TRP

19

TGG

—

27/13

LEU

TTA

GLY

19

GGG

—

27/13

LEU

TTA

ARG

19

AGG

—

27/13

LEU

TTA

THR

19

ATC

0.084 (1)

27/13

LEU

TTA

SER

19

TCC

—

27/13

LEU

TTA

ALA

19

GCG

0.01 (1)

28/14

LYS

AAG

ARG

19

CGG

0.42 ± 0.07 (2)

28/14

LYS

AAG

LEU

19

TTG

—

28/14

LYS

AAG

TRP

19

TGG

—

29/14

LYS

AAG

GLN

19

CAG

0.27 (1)

28/14

LYS

AAG

GLY

19

GGC

0.36 ± 0.07 (2)

29/14

LYS

AAG

PRO

19

CCC

0.10 ± 0.04 (2)

28/14

LYS

AAG

VAL

19

GTG

0.19 ± 0.12 (2)

29/15

GLN

CAG

ASN

19

AAC

1.62 ± 1.7 (3)

29/15

GLN

CAG

LEU

19

CTG

0.284

29/15

GLN

CAG

PRO

19

CCG

—

29/15

ARG

CAG

ARG

19

AGG

0.44 ± 0.16 (4)

29/15

GLN

CAG

VAL

19

GTG

0.62 ± 0.40 (4)

30/16

PRO

CCA

HIS

19

CAC

0.26 (1)

30/16

PRO

CCA

THR

19

ACG

0.36 (1)

30/16

PRO

CCA

GLY

19

GGG

1.2 ± 0.8 (3)

30/16

PRO

CCA

ASP

19

GAC

—

30/16

PRO

CCA

GLN

19

CAG

0.61 ± 0.37 (3)

30/16

PRO

CCA

SER

19

TCG

—

30/16

PRO

CCA

LEU

19

TTC

—

30/16

PRO

CCA

LYS

19

AAG

—

31/17

PRO

CCT

ASP

19

GAC

0.66 ± 0.71 (3)

36/22

ASP

GAC

VAL

19

GTG

—

37/23

PHE

TTC

SER

19

AGC

0.62 ± 0.40 (4)

37/23

PHE

TTC

PRO

19

CCG

0.65 ± 0.39 (4)

37/23

PHE

TTC

TRP

19

TGG

—

37/23

PHE

TTC

ILE

19

ATC

0.1 (1)

36/24

ASN

AAC

ALA

19

GCN

1.9 (1)

40/26

LEU

CTC

TRP

19

TGG

—

40/26

LEU

CTC

ARG

19

CGC

—

41/27

ASN

AAT

CYS

19

TGC

0.18 (1)

41/27

ASN

AAT

ARG

19

CGC

0.13 ± 0.13 (2)

41/27

ASN

AAT

LEU

19

CTG

0.09 ± 0.07 (2)

41/27

ASN

AAT

HIS

19

CAC

0.49 ± 0.26 (4)

41/27

ASN

AAT

MET

19

ATG

0.30 ± 0.38 (4)

41/27

ASN

AAT

PRO

19

CCG

0.12 (1)

42/28

GLY

GGG

ASP

19

GAC

5.7 ± 5.7 (6)

42/28

GLY

GGG

SER

19

AGC

4.3 ± 4.8 (7)

42/28

GLY

GGG

CYS

19

TGC

0.53 (1)

42/28

GLY

GGG

ALA

19

GCC

5.9 ± 4.1 (7)

43/29

GLU

GAA

ASN

19

AAC

0.050 (1)

43/29

GLU

GAA

TYR

19

TAC

0.010 (1)

43/29

GLU

GAA

LEU

19

CTC

<0.009 (1)

43/29

GLU

GAA

PHE

19

TTC

<0.009 (1)

43/29

GLU

GAA

ASP

19

GAC

0.044 (1)

43/29

GLU

GAA

ALA

19

GCC

<0.009 (1)

43/29

GLU

GAA

CYS

19

TGC

<0.009 (1)

43/29

GLU

GAA

SER

19

AGC

<0.009 (1)

44/30

ASP

GAC

SER

19

TCA

0.007 (1)

44/30

ASP

GAC

LEU

19

CTG

<0.007 (1)

44/30

ASP

GAC

ARG

19

AGG

<0.007 (1)

44/30

ASP

GAC

LYS

19

AAG

<0.007 (1)

44/30

ASP

GAC

THR

19

ACG

—

44/30

ASP

GAC

MET

19

ATG

<0.007 (1)

44/30

ASP

GAC

TRP

19

TGG

<0.007 (1)

44/30

ASP

GAC

PRO

19

CCC

<0.007 (1)

45/31

GLN

CAA

PRO

19

CCC

—

45/31

GLN

CAA

PHE

19

TTC

0.007 (1)

45/31

GLN

CAA

VAL

19

GTC

6.7 ± 6.1 (5)

45/31

GLN

CAA

MET

19

ATG

3.4 ± 1.8 (5)

45/31

GLN

CAA

LEU

19

TTG

1.1 ± 1.3 (2)

45/31

GLN

CAA

THR

19

ACG

0.96 ± 1.5 (3)

45/31

GLN

CAA

LYS

19

AAG

1.6 ± 2.2 (5)

45/31

GLN

CAA

TRP

19

TGG

0.10 (1)

46/32

ASP

GAC

PHE

19

TTC

1.2 ± 0.5 (3)

46/32

ASP

GAC

SER

19

TCC

7.9 ± 6.4 (4)

46/32

ASP

GAC

THR

19

ACC

1.8 ± 0.2 (2)

46/32

ASP

GAC

CYS

19

TGC

0.80 (1)

46/32

ASP

GAC

GLY

19

GGC

0.25 (1)

47/33

ILE

ATT

GLY

19

GGC

<0.015 (1)

47/33

ILE

ATT

VAL

19

GTG

0.38 (1)

47/33

ILE

ATT

HIS

19

CAC

0.10 (1)

47/33

ILE

ATT

SER

19

TCC

0.03 (1)

47/33

ILE

ATT

ARG

19

AGG

0.09 (1)

47/33

ILE

ATT

PRO

19

CCG

<0.015 (1)

48/34

LEU

CTG

SER

19

AGC

<0.009 (1)

48/34

LEU

CTG

CYS

19

TCG

—

48/34

LEU

CTG

ARG

19

CGC

<0.009 (1)

48/34

LEU

CTG

ILE

19

ATC

0.036 (1)

48/34

LEU

CTG

HIS

19

CAC

<0.009 (1)

48/34

LEU

CTG

PHE

19

TTC

<0.009 (1)

48/34

LEU

CTG

ASN

19

AAC

<0.009 (1)

49/35

MET

ATG

ARG

19

CGC

0.007 (1)

49/35

MET

ATG

ALA

19

GCC

0.091 (1)

49/35

MET

ATG

GLY

19

GGC

0.036 (1)

49/35

MET

ATG

PRO

19

CCC

<0.009 (1)

49/35

MET

ATG

ASN

19

AAC

0.23 (1)

49/35

MET

ATG

HIS

19

CAC

<0.009 (1)

49/35

MET

ATG

ASP

19

GAC

0.28 ± 0.48 (3)

50/36

GLU

GAA

LEU

19

CTC

0.01 (1)

50/36

GLU

GAA

THR

19

ACC

0.20 (1)

50/36

GLU

GAA

ASP

19

GAC

—

50/36

GLU

GAA

TYR

19

TAC

0.09 (1)

50/36

GLU

GAA

GLN

19

CTG

0.02 (1)

51/37

ASN

AAT

ARG

19

CGC

2.0 ± 0.8 (3)

51/37

ASN

AAT

MET

19

ATG

0.75 ± 0.50 (2)

51/37

ASN

AAT

PRO

19

CCG

2.77 ± 1.6 (3)

51/37

ASN

AAT

SER

19

TCC

0.87 ± 0.44 (3)

51/37

ASN

AAT

THR

19

ACG

2.3 ± 1.6 (3)

51/37

ASN

AAT

HIS

19

CAC

1.3 ± 0.9 (5)

52/38

ASN

AAC

HIS

19

CAC

0.004 (1)

52/39

ASN

AAC

ARG

19

CGC

0.004 (1)

52/36

ASN

AAC

LEU

19

TGG

0.003 (1)

52/38

ASN

AAC

GLY

19

GGC

0.22 (1)

52/38

ASN

AAC

SER

19

AGC

0.07 (1)

52/38

ASN

AAC

THR

19

ACG

0.44 ± 0.30 (3)

53/39

LEU

CTT

THR

19

ACC

<0.005 (1)

53/39

LEU

CTT

ALA

19

GCG

—

53/39

LEU

CTT

GLY

19

GGC

<0.005 (1)

53/39

LEU

CTT

GLU

19

GAG

<0.005 (1)

53/39

LEU

CTT

PRO

19

CCG

<0.005 (1)

53/39

LEU

CTT

LYS

19

AAG

<0.005 (1)

53/39

LEU

CTT

SER

19

AGC

0.008 (1)

53/39

LEU

CTT

MET

19

ATG

0.31 (1)

54/40

ARG

CGA

ASP

19

GAC

<0.005 (1)

54/40

ARG

CGA

ILE

19

ATC

0.05 (1)

54/40

ARG

CGA

SER

19

TCC

0.10 (1)

54/40

ARG

CGA

VAL

19

GTC

<0.005 (1)

54/40

ARG

CGA

THR

19

ACC

0.015 (1)

54/40

ARG

CGA

GLN

19

CAG

0.04 (1)

54/40

ARG

CGA

LEU

19

TTG

0.03 (1)

55/41

ARG

AGC

THR

19

ACC

0.65 ± 1.1 (4)

55/41

ARG

AGG

VAL

19

GTC

0.96 ± 0.36 (3)

55/41

ARG

AGG

SER

19

TCG

0.065 (1)

55/41

ARG

AGG

LEU

19

CTG

1.1 ± 1.2 (4)

55/41

ARG

AGC

GLY

19

GGC

1.0 ± 0.6 (4)

56/42

PRO

CCA

GLY

19

GGC

1.1 ± 0.8 (3)

56/42

PRO

CCA

CYS

19

TGC

0.21 (1)

56/42

PRO

CCA

SER

19

AGC

1.4 ± 0.4 (2)

56/42

PRO

CCA

GLN

19

CAG

1.8 (1)

56/42

PRO

CCA

LYS

19

AAG

0.60 (1)

57/43

ASN

AAC

GLY

6

19

GGC

—

58/44

LEU

CTG

SER

19

ACC

<0.041 (1)

58/44

LEU

CTG

ASP

19

GAC

<0.041 (1)

58/44

LEU

CTG

ARG

19

CGG

<0.041 (1)

58/44

LEU

CTG

GLN

19

CAG

<0.041 (1)

58/44

LEU

CTG

VAL

19

GTC

<0.041 (1)

58/44

LEU

CTG

CYS

19

TGC

—

59/45

GLU

GAG

TYR

19

TAC

0.41 ± 0.37 (5)

59/45

GLU

GAG

HIS

19

CAC

0.38 ± 0.31 (2)

59/45

GLU

GAG

LEU

19

CTC

0.46 ± 0.36 (6)

59/45

GLU

GAG

PRO

19

CCC

—

59/45

GLU

GAG

ARG

19

CGC

0.15 (1)

60/46

ALA

GCA

SER

19

AGC

0.91 ± 0.55 (4)

60/46

ALA

GCA

PRO

19

CCC

—

60/46

ALA

GCA

TYR

19

TAC

<0.008 (1)

60/46

ALA

GCA

ASN

19

AAC

0.38 (1)

60/46

ALA

GCA

THR

19

ACG

0.21 (1)

61/47

PHE

TTC

ASN

19

AAC

—

61/47

PHE

TTC

GLU

19

GAG

<0.010 (1)

61/47

PHE

TTC

PRO

19

CCC

—

61/47

PHE

TTC

LYS

19

AAG

<0.010 (1)

61/47

PHE

TTC

ARG

19

CGC

0.006 (1)

61/47

PHE

TTC

SER

19

TCG

0.17 (1)

62/48

ASN

AAC

HIS

19

CAC

—

62/48

ASN

AAC

VAL

19

GTG

0.37 ± 0.25 (4)

62/48

ASN

AAC

ARG

19

AGG

—

62/48

ASN

AAC

PRO

7

19

CCG

1.6 ± 0.4 (3)

62/48

ASN

AAC

PRO

19

CCG

2.0 ± 0.3 (3)

62/48

ASN

AAC

THR

8

19

ACG

2.3 ± 1.1 (3)

62/45

ASN

AAC

ASP

19

GAC

—

62/48

ASN

AAC

ILE

19

ATC

0.56 ± 0.24 (4)

63/49

ARG

A(G/A)G

TYR

19

TAC

0.47 (1)

63/49

ARG

A(G/A)G

TRP

19

TGG

0.09 (1)

63/49

ARG

A(G/A)G

LYS

19

AGG

0.52 (1)

63/49

ARG

A(G/A)G

SER

9

19

TCC

0.13 (1)

63/49

ARG

A(G/A)G

HIS

19

CAC

0.42 ± 0.25 (7)

63/49

ARG

A(G/A)G

PRO

19

CCG

<0.014 ± 0.013(2)

63/49

ARG

A(G/A)G

VAL

19

GTG

0.39 ± 0.34 (3)

64/50

ALA

GCT

ASN

19

AAC

1.5 ± 2.9 (4)

64/50

ALA

GCT

PRO

19

CCG

<0.023 (1)

64/50

ALA

GCT

SER

19

AGC

<0.023 (1)

64/50

ALA

GCT

LYS

19

AAG

<0.047 (1)

65/51

VAL

GTC

THR

19

ACC

0.71 ± 0.64 (3)

65/51

VAL

GTC

PRO

19

CCG

<0.014 (1)

65/51

VAL

GTC

HIS

19

CAC

<0.014 (1)

65/51

VAL

GTC

LEU

19

CTC

0.42 (1)

65/51

VAL

GTC

PHE

19

TTC

0.061 (1)

65/51

VAL

GTC

SER

19

TCC

0.34 (1)

66/52

LYS

AAG

ILE

10

19

ATC

0.42 (1)

66/52

LYS

AAG

ARG

19

AGG

0.79 ± 0.18 (2)

66/52

LYS

AAG

VAL

19

GTC

0.38 ± 0.17 (2)

66/52

LYS

AAG

ASN

19

AAC

0.32 (1)

66/52

LYS

AAG

GLU

19

GAG

0.14 (1)

66/52

LYS

AAG

SER

19

TCG

0.31 (1)

66/52

LYS

AAG

VAL

11

19

GTG

0.055 (1)

67/53

SER

AGT

ALA

19

GCG

<0.014 (1)

67/53

SER

AGT

PHE

19

TTC

1.2 ± 0.2 (2)

67/53

SER

AGT

VAL

19

GTG

0.24 (1)

67/53

SER

AGT

GLY

19

GGG

0.50 ± 0.29 (4)

67/53

SER

AGT

ASN

19

AAC

0.52 ± 0.28 (7)

67/53

SER

AGT

ILE

19

ATC

0.29 (1)

67/53

SER

AGT

PRO

19

CCG

0.055 (1)

67/53

SER

AGT

HIS

19

CAC

0.99 ± 0.62 (6)

68/54

LEU

TTA

VAL

19

GTC

0.14 (1)

68/54

LEU

TTA

TRP

19

TGG

0.07 (1)

68/54

LEU

TTA

SER

19

AGC

<0.003 (1)

68/54

LEU

TTA

ILE

19

ATC

0.84 ± 0.47 (3)

68/54

LEU

TTA

PHE

19

TTC

1.7 ± 0.3 (3)

68/54

LEU

TTA

THR

19

ACG

0.011 (1)

68/54

LEU

TTA

HIS

19

CAC

0.82 ± 0.45 (2)

69/55

GLN

CAG

ALA

19

GCG

1.2 ± 0.8 (3)

69/55

GLN

CAG

PRO

19

CCA

0.74 0.45 (4)

69/55

GLN

CAG

THR

19

ACG

0.97 ± 0.46 (4)

69/55

GLN

CAG

TRP

19

TGG

—

69/55

GLN

CAG

GLU

19

GAG

1.4 ± 0.7 (3)

69/55

GLN

CAG

ARG

19

CGG

1.4 ± 1.1 (3)

69/55

GLN

CAG

GLY

19

GGG

0.68 ± 0.02 (2)

69/55

GLN

CAG

LEU

19

CTC

—

70/56

ASN

AA(C/T)

LEU

19

TTG

0.032 (1)

70/56

ASN

AA(C/T)

VAL

19

GTG

—

70/56

ASN

AA(C/T)

TRP

19

TGG

—

70/56

ASN

AA(C/T)

PRO

12

19

CCG

0.43 ± 0.29 (2)

70/56

ASN

AA(C/T)

ALA

13

19

GCC

0.03 (1)

71/57

ALA

GCA

MET

19

ATG

0.23 (1)

71/57

ALA

GCA

LEU

19

CTG

<0.005 (1)

71/57

ALA

GCA

PRO

19

CCC

0.58 (1)

71/57

ALA

GCA

ARG

19

AGG

0.66 (1)

71/57

ALA

GCA

GLU

19

GAG

0.46 ± 0.27

71/57

ALA

GCA

THR

19

ACC

0.34 ± 0.41 (3)

71/57

ALA

GCA

GLN

19

GGC

0.42 ± 0.32 (3)

71/57

ALA

GCA

TRP

19

TGG

—

71/57

ALA

GCA

ASN

19

AAC

0.09 (1)

72/58

SER

TCA

GLU

19

GAG

0.62 ± 0.27 (3)

72/56

SER

TCA

MET

19

ATG

0.45 ± 0.55 (3)

72/58

SER

TCA

ALA

19

GCC

0.48 ± 0.33 (3)

72/58

SER

TCA

HIS

19

CAC

0.10 (1)

72/58

SER

TCA

ASN

19

AAC

0.38 ± 0.44 (3)

72/58

SER

TCA

ARG

19

CGG

0.81 ± 0.43 (4)

72/58

SER

TCA

ASP

19

GAC

0.58 ± 0.39 (3)

73/59

ALA

GCA

GLU

19

GAG

0.49 ± 0.32 (3)

73/59

ALA

GCA

ASP

19

GAC

0.27 (1)

73/59

ALA

GCA

LEU

19

CTG

0.55 ± 0.45 (4)

73/59

ALA

GCA

SER

19

AGC

0.37 ± 0.36 (2)

73/59

ALA

GCA

GLY

19

CGG

0.38 ± 0.32 (3)

73/59

ALA

GCA

THR

19

ACC

0.31 (1)

73/59

ALA

GCA

ARG

19

AGG

0.40 ± 0.18 (3)

74/60

ILE

AT(T/C)

MET

19

ATG

<0.16 (1)

74/60

ILE

AT(T/C)

THR

19

ACG

—

74/60

ILE

AT(T/C)

PRO

19

CCG

—

74/60

ILE

AT(T/C)

ARG

19

AGG

—

74/60

ILE

AT(T/C)

GLY

19

GCG

0.006 (1)

74/60

ILE

AT(T/C)

ALA

19

GCG

—

75/61

GLU

GAG

LYS

19

AAG

0.07 ± 0.07 (2)

75/61

GLU

GAG

GLY

19

GGG

0.27 ± 0.20 (2)

75/61

GLU

GAG

ASP

19

GAC

0.18 (1)

75/61

GLU

GAG

PRO

19

CCG

—

75/61

GLU

GAG

TRP

19

TGG

—

75/61

GLU

GAG

ARG

19

CGG

—

75/61

GLU

GAG

SER

19

TCG

0.27 ± 0.22 (3)

75/61

GLU

GAG

GLN

19

CAG

0.40 ± 0.38 (3)

75/61

GLU

GAG

LEU

19

TTG

—

76/62

SER

AGC

VAL

19

GTG

1.0 ± 0.2 (2)

76/62

SER

AGC

ALA

19

GCG

0.94 ± 0.46 (2)

76/62

SER

AGC

ASN

19

AAC

1.2 (1)

76/62

SER

AGC

TRP

19

TGG

—

76/62

SER

AGC

GLU

19

GAG

0.90 ± 0.19 (2)

76/62

SER

AGC

PRO

19

CCG

2.1 ± 0.8 (4)

76/62

SER

AGC

GLY

19

GGC

1.3 ± 1.0 (4)

76/62

SER

AGC

ASP

19

GAC

0.29 (1)

77/63

ILE

ATT

SER

19

AGC

0.48 ± 0.38 (4)

77/63

ILE

ATT

ARG

19

CGC

0.09 ± 0.04 (2)

77/63

ILE

ATT

THR

19

ACG

<0.008 (1)

77/63

ILE

ATT

LEU

19

TTG

2.0 ± 0.1 (3)

78/64

LEU

CTT

ALA

19

GCG

—

78/64

LEU

CTT

SER

19

TCC

—

78/64

LEU

CTT

GLU

19

GAG

<0.006 (1)

78/64

LEU

CTT

PHE

19

TTC

—

78/64

LEU

CTT

GLY

19

GGG

—

78/64

LEU

CTT

ARG

19

AGG

—

79/65

LYS

AA(A/G)

THR

19

ACA

0.77 ± 0.91 (6)

79/65

LYS

AA(A/G)

GLY

19

GGG

1.1 ± 0.9 (6)

79/65

LYS

AA(A/G)

ASN

19

AAC

1.0 ± 0.6 (6)

79/65

LYS

AA(A/G)

MET

19

ATG

1.6 ± 0.7 (6)

79/65

LYS

AA(A/G)

ARG

19

CGC

1.04 ± 0.7 (7)

79/65

LYS

AA(A/G)

ILE

19

ATC

1.0 ± 0.6 (6)

79/65

LYS

AA(A/G)

GLY

19

GGG

1.2 ± 0.4 (6)

79/65

LYS

AA(A/G)

ASP

19

GAC

0.72 ± 0.38 (7)

80/66

ASN

AAT

TRP

19

TGG

—

80/66

ASN

AAT

VAL

19

GTC

0.32 (1)

80/66

ASN

AAT

GLY

19

GGC

1.5 ± 1.4 (4)

80/66

ASN

AAT

THR

19

ACG

0.13 (1)

80/66

ASN

AAT

LEU

19

CTG

0.33 ± 0.14 (2)

80/66

ASN

AAT

GLU

19

GAG

1.1 ± 0.8 (4)

80/66

ASN

AAT

ARG

19

AGG

1.0 ± 0.8 (4)

81/67

LEU

CTC

GLN

19

CAA

—

81/67

LEU

CTC

GLY

19

GGC

<0.023 (1)

81/67

LEU

CTC

ALA

19

GCG

<0.047 (1)

81/67

LEU

CTC

TRP

19

TGG

<0.005 (1)

81/67

LEU

CTC

ARG

19

CGG

—

81/67

LEU

CTC

VAL

19

GTG

0.16 ± 0.18 (2)

81/67

LEU

CTC

LYS

19

AAG

—

82/68

LEU

C(TG/CC)

GLN

19

CAG

1.8 ± 0.3 (3)

82/68

LEU

C(TG/CC)

LYS

19

AAG

0.05 (1)

82/68

LEU

C(TG/CC)

TRP

19

TGG

2.7 ± 1.3 (4)

82/68

LEU

C(TG/CC)

ARG

19

AGC

1.1 ± 0.2 (3)

82/68

LEU

C(TG/CC)

ASP

19

GAC

2.7 ± 1.3 (4)

82/68

LEU

C(TG/CC)

VAL

19

GTG

1.5 ± 1.1 (5)

83/69

PRO

CCA

ALA

19

GCA

0.41 (1)

83/69

PRO

CCA

THR

19

ACC

0.66 ± 0.12 (3)

83/69

PRO

CCA

ARG

19

CGG

—

83/69

PRO

CCA

TRP

19

TGG

0.29 (1)

83/69

PRO

CCA

MET

19

ATG

0.43 ± 0.28 (3)

84/70

CYS

TG(T/C)

GLU

19

GAG

<0.014 (1)

84/70

CYS

TG(T/C)

GLY

19

GGG

<0.006 (1)

84/70

CYS

TG(T/C)

ARG

19

AGG

—

84/70

CYS

TG(T/C)

MET

19

ATG

—

84/70

CYS

TG(T/C)

VAL

19

GTG

—

85/71

LEU

CTG

ASN

19

AAC

—

85/71

LEU

CTG

VAL

19

GTG

0.52 ± 0.21 (5)

85/71

LEU

CTG

GLN

19

CAG

—

86/72

PRO

CCC

CYS

19

TGC

—

86/72

PRO

CCC

ARG

19

AGG

—

86/72

PRO

CCC

ALA

19

GCG

—

86/72

PRO

CCC

LYS

19

AAG

—

87/73

LEU

(C/A)TG

SER

19

AGC

1.5 ± 0.4 (3)

87/73

LEU

(C/A)TG

TRP

19

TGG

—

87/73

LEU

(C/A)TG

GLY

19

GGG

—

88/74

ALA

GCC

LYS

19

AAG

—

88/74

ALA

GCC

ARG

19

AGG

0.11 ± 0.10 (2)

88/74

ALA

GCC

VAL

19

GTG

0.09 ± 0.02 (2)

88/74

ALA

GCC

TRP

19

TGG

1.8 ± 0.2 (2)

89/75

THR

AC(G/A)

ASP

19

GAC

0.24 ± 0.10 (2)

89/75

THR

AC(G/A)

CYS

19

TGC

—

89/75

THR

AC(G/A)

LEU

19

CTC

0.01 (1)

89/75

THR

AC(G/A)

VAL

19

GTG

0.08 (1)

69/75

THR

AC(G/A)

GLU

19

GAG

0.11 (1)

89/75

THR

AC(G/A)

HIS

19

CAC

0.16 ± 0.06 (2)

89/75

THR

AC(G/A)

ASN

19

AAC

0.21 ± 0.04 (2)

89/75

THR

AC(G/A)

SER

19

TCG

0.25 ± 0.07 (2)

90/76

ALA

GCC

PRO

19

CCC

0.03 (1)

90/76

ALA

GCC

SER

19

TCG

—

90/76

ALA

GCC

THR

19

ACC

0.48 (1)

90/76

ALA

GCC

GLY

19

GGC

<0.006 (1)

90/76

ALA

GCC

ASP

19

GAC

0.44 ± 0.29 (4)

90/76

ALA

GCC

ILE

19

ATC

—

90/76

ALA

GCC

MET

19

ATG

0.25 ± 0.13 (2)

91/77

ALA

GCA

PRO

19

CCC

1.9 ± 1.2 (3)

91/77

ALA

GCA

SER

19

TCC

0.12 ± 0.07 (2)

91/77

ALA

GCA

THR

19

ACC

0.48 ± 0.16 (2)

91/77

ALA

GCA

PHE

19

TTC

0.44 ± 0.50 (3)

91/77

ALA

GCA

LEU

19

CTC

0.43 ± 0.27 (S)

91/77

ALA

GCA

ASP

19

GAC

0.55 ± 0.09 (2)

91/77

ALA

GCA

HIS

19

CAC

—

92/76

PRO

CCC

PHE

19

TTC

—

92/78

PRO

CCC

ARG

19

CGG

—

92/78

PRO

CCC

SER

19

AGC

0.26 (1)

92/78

PRO

CCC

LYS

19

AAG

—

92/78

PRO

CCC

HIS

19

CAC

—

92/78

PRO

CCC

LEU

19

CTG

—

93/79

THR

ACG

ASP

19

GAC

1.3 ± 0.7 (4)

93/79

THR

ACG

SER

19

TCG

0.70 ± 0.56 (4)

93/79

THR

ACG

ASN

19

AAC

—

93/79

THR

ACG

PRO

19

CCC

0.53 ± 0.36 (4)

93/79

ThR

ACG

ALA

19

GCG

1.13 ± 0.2 (3)

93/79

THR

ACG

LEU

19

CTG

0.69 ± 0.42

93/79

THR

ACG

ARG

19

CGC

0.93 ± 0.96 (4)

94/80

ARG

CGA

ILE

19

ATC

<0.020 (1)

94/80

ARG

CGA

SER

19

TCC

<0.100 (1)

94/80

ARG

CGA

GLU

19

GAG

<0.020 (1)

94/90

ARG

CGA

LEU

19

CTG

<0.020 (1)

94/50

ARG

CGA

VAL

19

GTG

<0.024 (1)

94/80

ARG

CGA

PRO

19

CCC

<0.024 (1)

95/81

HIS

CAT

GLN

19

CAG

<0.010 (1)

95/81

HIS

CAT

PRO

19

CCG

1.6 ± 0.8 (3)

95/81

HIS

CAT

ARG

19

CGC

4.7 ± 5.9 (2)

95/81

HIS

CAT

VAL

19

GTC

1.2 ± 1.7 (2)

95/81

HIS

CAT

LEU

19

CTC

0.7 (1)

95/81

HIS

CAT

GLY

19

GGC

1.7 ± 2.4 (5)

95/81

HIS

CAT

THR

19

ACC

2.9 ± 4.5 (4)

95/81

HIS

CAT

TYR

19

TAC

0.07 (1)

96/82

PRO

CCA

LYS

19

AAG

<0.010 ± 0.001 (2)

96/82

PRO

CCA

TYR

19

TAC

0.69 (1)

96/82

PRO

CCA

GLY

19

GGG

<0.040 (1)

96/82

PRO

CCA

ILE

19

ATC

<0.040 (1)

96/82

PRO

CCA

THR

19

ACC

<0.040 (1)

97/83

ILE

ATC

VAL

19

GTC

0.91 ± 1.2 (8)

97/83

ILE

ATC

LYS

19

AAG

<0.024

97/83

ILE

ATC

ALA

19

GCG

0.15 (1)

97/83

ILE

ATC

ASN

19

AAT

<0.02 (1)

98/84

HIS

CAT

ILE

19

ATC

5.0 ± 4.9 (12)

98/84

HIS

CAT

ASN

19

AAC

1.4 ± 0.4 (2)

98/84

HIS

CAT

LEU

19

CTC

2.4 ± 1.0 (2)

98/84

HIS

CAT

ASP

19

GAC

0.38 ± 0.49 (5)

98/84

HIS

CAT

ALA

19

GCC

2.0 ± 1.0 (3)

98/84

HIS

CAT

THR

19

ACG

1.6 ± 0.3 (2)

98/84

HIS

CAT

LEU

19

TTG

1.5 (1)

98/84

HIS

CAT

PRO

19

CCG

0.55 (1)

99/85

ILE

ATC

LEU

19

CTG

1.4 ± 1.4 (7)

99/85

ILE

ATC

ARG

19

CGC

<0.025 (1)

99/85

ILE

ATC

ASP

19

GAC

<0.025 (1)

99/65

ILE

ATC

VAL

19

GTC

0.51 ± 0.59 (3)

99/55

ILE

ATC

PRO

19

CCG

<0.025 (1)

99/85

ILE

ATC

GLN

19

CAG

<0.018 ± 0.010 (2)

99/85

ILE

ATC

GLY

19

GGG

<0.018 ± 0.10 (2)

99/85

ILE

ATC

SER

19

TCG

<0.025 (1)

99/85

ILE

ATC

PHE

19

TTC

0.45 (1)

99/85

ILE

ATC

HIS

19

CAC

<0.025 (1)

100/86

LYS

AAG

TYR

19

TAC

0.03 (1)

100/96

LYS

AAG

LEU

19

TTG

0.33 ± 0.31 (3)

100/96

LYS

AAG

HIS

19

CAC

0.36 ± 0.22 (9)

100/86

LYS

AAG

ARG

19

AGC

4.7 ± 5.9 (4)

100/86

LYS

AAG

ILE

19

ATC

0.95 (1)

100/86

LYS

AAG

SER

19

AGC

0.95 (1)

100/86

LYS

AAG

GLN

19

CAG

0.78 ± 0.80 (7)

100/86

LYS

AAG

PRO

19

CCG

0.70 (1)

101/87

ASP

GAC

PRO

19

CCC

2.3 ± 3.1 (4)

101/87

ASP

GAC

MET

19

ATG

1.8 ± 2.5 (6)

101/87

ASP

GAC

LYS

19

AAG

1.2 ± 1.7 (3)

101/87

ASP

GAC

HIS

19

CAC

2.5 (1)

101/87

ASP

GAC

THR

19

ACG

0.90 ± 0.77 (3)

101/87

ASP

GAC

TYR

19

TAC

0.59 (1)

101/87

ASP

GAC

VAL

19

GTC

0.42 (1)

101/87

ASP

GAC

TYR

19

TAC

1.0 ± 0.02 (2)

101/87

ASP

GAC

GLN

19

CAG

0.07 (1)

102/88

GLY

GGT

LEU

19

CTC

<0.015 ± 0.007 (2)

102/88

GLY

GGT

GLU

19

GAG

0.40 ± 0.07 (3)

102/88

GLY

GGT

LYS

19

AGG

0.16 ± 0.14 (2)

102/88

GLY

GGT

SER

19

TCC

0.29 (1)

102/88

GLY

GGT

TYR

19

TAC

0.04 (1)

102/88

GLY

GGT

PRO

19

CCC

<0.011 (1)

103/89

ASP

GAC

SER

19

TCC

0.02 (1)

104/90

TRP

TGG

VAL

19

GTG

0.11 ± 0.06 (5)

104/90

TRP

TGG

CYS

19

AGC

0.07 ± 0.03 (5)

104/90

TRP

TGG

TYR

19

TAC

0.34 ± 0.42 (5)

104/90

TRP

TGG

THR

19

ACC

0.04 ± 0.02 (2)

104/90

TRP

TGG

MET

19

ATG

0.14 (1)

104/90

TRP

TGG

PRO

19

CCC

0.02 ± 0.02 (2)

104/90

TRP

TGG

LEU

19

TTG

0.65 ± 1.0 (3)

104/90

TRP

TGG

GLN

19

CAG

0.008 (1)

104/90

TRP

TGG

LYS

19

AAG

—

104/90

TRP

TGG

GLY

19

GAG

—

104/90

TRP

TGG

ALA

19

GCC

—

104/90

TRP

TGG

PHE

19

TTC

—

104/90

TRP

TGG

GLY

19

GGC

—

105/91

ASN

AAT

PRO

19

CCG

4.8 ± 8.5 (5)

105/91

ASN

AAT

ALA

19

GCC

0.65 ± 0.30 (3)

105/91

ASN

AAT

PHE

19

TTC

0.13 (1)

105/91

ASN

AAT

SER

19

TCC

1.9 ± 2.7 (5)

105/91

ASN

AAT

TRP

19

TGG

0.95 (1)

105/91

ASN

AAT

GLN

19

CAA

0.57 ± 0.52 (3)

105/91

ASN

AAT

TYR

19

TAC

0.66 ± 0.53 (4)

105/91

ASN

AAT

LEU

19

CTC

0.87 ± 0.79 (2)

105/91

ASN

AAT

LYS

19

AAG

0.70 (1)

105/91

ASN

AAT

ILE

19

ATC

1.0 (1)

105/91

ASN

AAT

ASP

19

GAC

1.0 ± 0.9 (4)

105/91

ASN

AAT

HIS

19

CAC

0.71 ± 0.48 (2)

106/92

GLU

GAA

SER

19

TCC

0.17 ± 0.21 (2)

106/92

GLU

GAA

ALA

19

GCG

0.235 ± 0.26 (2)

106/92

GLU

GAA

LYS

19

AAG

—

106/92

GLU

GAA

THR

19

ACC

—

106/92

GLU

GAA

ILE

19

ATC

—

106/92

GLU

GAA

GLY

19

GGC

0.70 ± 0.76 (4)

106/92

GLU

GAA

PRO

19

CCC

—

108/94

ARG

CGG

LYS

19

AAG

0.11 ± 0.03 (2)

108/94

ARG

CGG

ASP

19

GAC

—

108/94

ARG

CGG

LEU

19

TTG

0.01 (1)

108/94

ARG

CGG

THR

19

ACG

0.08 (1)

108/94

ARG

CGG

ILE

19

ATC

<0.01 (1)

108/94

ARG

CGG

PRO

19

CCC

—

109/95

ARG

AGG

THR

19

ACC

1.1 ± 0.2 (3)

109/95

ARG

AGG

PRO

19

CCC

—

109/95

ARG

AGG

GLU

19

GAG

1.1 ± 0.1 (3)

109/95

ARG

AGG

TYR

19

TAC

<0.006 (1)

109/95

ARG

AGG

LEU

19

CTC

1.2 ± 0.9 (4)

109/95

ARG

AGG

SER

19

TCG

1.7 ± 0.8 (4)

109/95

ARG

AGG

GLY

19

GGG

0.17 (1)

110/96

LYS

AAA

ALA

19

GCC

<0.08 (1)

110/96

LYS

AAA

ASN

19

AAC

—

110/96

LYS

AAA

THR

19

ACG

—

110/96

LYS

AAA

LEU

19

CTC

—

110/96

LYS

AAA

ARG

19

CGG

—

110/96

LYS

AAA

GLN

19

CAG

—

110/96

LYS

AAA

TRP

19

TGG

—

111/97

LEU

CTG

ILE

19

ATC

—

111/97

LEU

CTG

ARG

19

CGG

—

111/97

LEU

CTG

ASP

19

GAC

—

111/97

LEU

CTG

MET

19

ATG

—

112/98

THR

ACG

VAL

19

GTG

0.55 ± 0.44 (3)

112/98

THR

ACG

GLN

19

CAG

1.7 ± 1.0 (3)

112/98

THR

ACG

TYR

19

TAC

<0.018 (1)

112/98

THR

ACG

GLU

19

GAG

0.12 (1)

112/98

THR

ACG

HIS

19

CAC

0.25 ± 0.40 (3)

112/99

THR

ACG

SER

19

TCC

0.17 ± 0.15 (2)

112/98

THR

ACG

PHE

19

TTC

—

113/99

PHE

TTC

SER

19

AGC

—

113/99

PHE

TTC

CYS

19

TGC

—

113/99

PHE

TTC

HIS

19

CAC

<0.009 (1)

113/99

PHE

TTC

GLY

19

GGC

—

113/99

PHE

TTC

TRP

19

TGG

—

113/99

PHE

TTC

TYR

19

TAC

0.07 (1)

113/99

PHE

TTC

ASN

19

AAC

—

114/100

TYR

TAT

CYS

19

TGC

—

114/100

TYR

TAT

HIS

19

CAC

—

114/100

TYR

TAT

SER

19

AGC

—

114/100

TYR

TAT

TRP

19

TGG

0.88 (1)

114/100

TYR

TAT

ARG

19

AGG

—

114/100

TYR

TAT

LEU

19

CTC

<0.018 (1)

115/101

LEU

CTG

ASN

19

AAC

<0.004 (1)

115/101

LEU

CTG

VAL

19

GTG

—

115/101

LEU

CTG

PRO

19

CCC

<0.004 (1)

115/101

LEU

CTG

ARG

19

AGG

<0.004 (1)

115/101

LEU

CTG

ALA

19

GCG

0.50 (1)

115/101

LEU

CTG

HIS

19

CAC

—

115/101

LEU

CTG

THR

19

ACC

—

115/101

LEU

CTG

TRP

19

TGG

—

115/101

LEU

CTG

MET

19

ATG

<0.008 (1)

116/102

LYS

AAA

LEU

14

19

TTG

—

116/102

LYS

AAA

PRO

14

19

CCG

<0.004 (1)

116/102

LYS

AAA

THR

14

19

ACC

0.50 (1)

116/102

LYS

AAA

MET

14

19

ATG

0.13 (1)

116/102

LYS

AAA

ASP

14

19

GAC

<0.018 (1)

116/102

LYS

AAA

VAL

19

GTG

2.3 ± 1.2 (5)

116/102

LYS

AAA

GLU

19

GAG

0.06 (1)

116/102

LYS

AAA

ARG

19

CGC

0.06 (1)

116/102

LYS

AAA

TRP

19

TGG

2.3 ± 1.0 (4)

116/102

LYS

AAA

SER

19

TCG

0.69 ± 0.51 (5)

116/102

LYS

AAA

LEU

19

CTC

0.14 ± 0.02 (2)

116/102

LYS

AAA

ILE

19

ATC

1.3 ± 0.3 (3)

116/102

LYS

AAA

THR

19

ACG

0.84 ± 0.30 (4)

117/103

THR

ACC

SER

19

AGC

1.1 ± 0.2 (3)

117/103

THR

ACC

ASN

19

AAC

0.31 ± 0.39 (3)

117/103

THR

ACC

ILE

19

ATC

—

117/103

THR

ACC

TRP

19

TGG

0.02 (1)

117/103

THR

ACC

LYS

19

AAG

<0.005 (1)

117/103

THR

ACC

PRO

19

CCG

—

118/104

LEU

CTT

SER

19

TCA

—

118/104

LEU

CTT

PRO

19

CCC

—

118/104

LEU

CTT

ALA

19

GCC

—

118/104

LEU

CTT

GLU

19

GAG

—

118/104

LEU

CTT

CYS

19

TGC

—

118/104

LEU

CTT

ASP

19

GAC

—

118/104

LEU

CTT

TYR

19

TAC

—

119/105

GLU

GAG

SER

19

TCC

0.26 ± 0.19 (2)

119/105

GLU

GAG

LYS

19

AAG

0.04 (1)

119/105

GLU

GAG

PRO

19

CCG

0.31 ± 0.27 (3)

119/105

GLU

GAG

LEU

19

CTG

0.35 ± 0.35 (3)

119/105

GLU

GAG

THR

19

ACC

0.25 ± 0.27 (3)

119/105

GLU

GAG

TYR

19

TAC

0.30 ± 0.32 (3)

119/105

GLU

GAG

ARG

19

CGC

0.06 (1)

120/106

ASN

AAT

ALA

19

GCC

<0.009 (1)

120/106

ASN

AAT

PRO

19

CCC

1.7 ± 0.7 (3)

120/106

ASN

AAT

LEU

19

TTG

1.2 ± 0.3 (3)

120/106

ASN

AAT

HIS

19

CAC

1.0 ± 0.3 (2)

120/106

ASN

AAT

VAL

19

GTG

1.7 ± 0.3 (3)

120/106

ASN

AAT

GLN

19

CAG

0.85 ± 0.16 (2)

121/107

ALA

GCG

SER

19

AGC

1.2 ± 0.2 (3)

121/107

ALA

GCG

ILE

19

ATC

2.8 ± 2.5 (2)

121/107

ALA

GCG

ASN

19

AAC

0.91 ± 0.77 (5)

121/107

ALA

GCG

PRO

19

CCG

1.3 (1)

121/107

ALA

GCG

LYS

19

AAG

0.26 ± 0.24 (2)

121/107

ALA

GCG

ASP

19

GAC

1.8* ± 0.9 (3)

121/107

ALA

GCG

GLY

19

GGC

0.69 (1)

122/108

GLN

GCG

SER

19

AGC

0.9.6 ± 0.41 (3)

122/108

GLN

CA(G/A)

MET

19

ATG

1.7 ± 0.5 (3)

122/108

GLN

CA(G/A)

TRP

19

TGG

1.4 (1)

122/108

GLN

CA(G/A)

ARG

19

AGG

0.78 (1)

122/108

GLN

CA(G/A)

PHE

19

TTC

2.3 ± 1.1 (3)

122/108

GLN

CA(G/A)

PRO

19

CCG

1.0 (1)

122/108

GLN

CA(G/A)

HIS

19

CAC

1.4 (1)

122/108

GLN

CA(G/A)

ILE

19

ATC

2.7 ± 0.8 (3)

122/108

GLN

CA(G/A)

TYR

19

TAC

1.7 ± 0.3 (2)

122/108

GLN

CA(G/A)

CYS

19

TGC

0.58 (1)

123/109

ALA

GCT

MET

19

ATG

2.0 ± 0.2 (3)

123/109

ALA

GCT

GLU

19

GAG

2.1 ± 1.0 (3)

123/109

ALA

GCT

HIS

19

CAC

0.98 ± 0.72 (3)

123/109

ALA

GCT

SER

19

AGC

1.4 ± 0.8 (3)

123/109

ALA

GCT

PRO

19

CCC

0.64 ± 0.16 (2)

123/109

ALA

GCT

TYR

19

TAC

0.51 ± 0.25 (2)

123/109

ALA

GCT

LEU

19

CTG

1.2 ± 0.1 (2)

1

The first position number representes the amino acid position in (1-133)hIL-3 and the second number represents the position in (15-125)hIL-3 in which the Asn at position 15 of native hIL-3 is position 1 in (15-125)hIL-3 (See the numbering for Formula XI)

2

Double mutant; has PRO at position 35.

3

Double mutant; has THR at position 49.

4

Double mutant; has Gly at position 32.

5

Double mutant; has Leu at position 31.

6

Double mutant; has Gly at position 46.

7

Double mutant; Arg at position 42.

8

Double mutant; Phe at position 53.

9

Double mutant; has Val at position 49.

10

Double mutant; has Pro at position 73.

11

Double mutant; has Thr at position 64.

12

Double mutant; has Pro at position 73.

13

Double mutant; has Met at position 74.

14

Double mutant; has Ser at position 105.

The mutants in Table 6 were made as described in the Examples, particularly Examples 19, 20, 21 and 38 to 53.

It will be apparent to those skilled in the art that other codons besides those shown in Table 6 can also code for the substituted amino acids in the hIL-3 muteins. The present invention includes the DNAs encoding the mutant hIL-3 polypeptides of the invention including the various codons which can code for the parental and substituted amino acids of the hIL-3 muteins of the invention due to the degeneracy of the genetic code.

hIL-3 (15-125) variant genes encoding the variants listed in Table 6 can also be expressed from intracellular expression vectors to produce large quantities of the variant protein which can be purified and assayed for biological activity. The hIL-3 variant genes, from Table 6, can be excised from the secretion expression vector, as a 345 base pair NcoI/HindIII fragment and ligated into an appropriate intracellular expression vector, such as pMON2341 digested with NcoI and HindIII. Examples of variants transferred to pMON2341 in this manner are shown in Table 7. Two examples of such a transfer are described. in the construction of pMON13215 (EXAMPLE 64) and pMON13252 (EXAMPLE 65).

EXAMPLE 64

Construction of pMON13215

Plasmid, pMON2341, DNA was digested with restriction enzymes NcoI and HindIII resulting in a 3619 base pair NcoI/HindIII fragment. The genetic elements derived from pMON2341 are the beta-lactamase gene (AMP), pBR327 origin of replication, F1 phage origin of replication as the transcription terminator, precA, g10L ribosome binding site. The plasmid encoding the hIL-3 (15-125) Trp

(116)

variant, from Table 6 wag digested with NcoT and HindIII resulting in a 345 base pair NcoI/HindIII fragment. The 345 Base pair NcoI/HindIII fragment was ligated with the 3619 base pair fragment from pMON2341 and the ligation reaction mixture was used to transform

E.coli

K-12 strain JM101. Plasmid DNA was isolated and screened by restriction anaylsis using NcoI and HindIII. Positive clones contained a 345 base pair NcoI/HindIII. This construct was designated PMON132I5. The plasmid, pMON13215, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

PEPTIDE A9; (15-125)HIL-3 TRP

(116)

PMON13215

Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu

[SEQ ID NO:217]

15 20 25

Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly

30 35 40

Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn

45 50 55

Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser

60 65 70

Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu

75 80 85

Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly

90 95 100

Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Trp Thr

105 110 115

Leu Glu Asn Ala Gln Ala Gln Gln

120 125

DNA sequence #A9 pMON13215 116w

ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA

[SEQ ID NO:220]

GCCACCGCTG CCGCTGCTGG ACTTCAACAA CCTCAATGGT GAAGACCAAG

ATATCCTGAT GGAAAATAAC CTTCGTCGTC CAAACCTCGA GGCATTCAAC

CGTGCTGTCA ACTCTCTGCA GAATGCATCA GCAATTGAGA GCATTCTTAA

AAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC ACGCGACATC

CAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACC

TTCTATCTGT GGACCTTGGA GAACGCGCAG GCTCAACAG

EXAMPLE 65

Construction of pMON13252

Plasmid, pMON2341, DNA was digested with restriction enzymes NcoI and HindIII resulting in a 3619 base pair NcoI/HindIII fragment. The genetic elements derived from pMON2341 are the beta-lactamase gene (AMP), pBR327 origin of replication F1 phage origin of replication as the transcription terminator, precA, g10L ribosome binding site. The plasmid encoding the hIL-3 (15-125) Asp

( 50)

variant, from Table 6, was digested with NcoI and HindIII resulting in a 345 base pair NcoI/HindIII fragment. This 345 Base pair NcoI/HindIII fragment was ligated with the 3619 base pair fragment from pMON2341 and the ligation reaction mixture was used to transform

E. coli

K-12 strain JM101. Plasmid DNA was isolated and screened by restriction analysis using NcoI and HindIII. Positive clones contained a 345 base pair NcoI/HindIII. This construct was designated pMON13252. The plasmid, pMON13252, encodes the (15-125) hIL-3 variant with the following amino acid sequence:

PEPTIDE A10; (15-125)HIL-3 ASP

(50)

pMON13252

Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu

[SEQ ID NO:218]

15 20 25

Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly

30 35 40

Glu Asp Gln Asp Ile Leu Met Asp Asn Asn Leu Arg Arg Pro Asn

45 50 55

Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser

60 65 70

Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu

75 80 85

Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly

90 95 100

Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr

105 110 115

Leu Glu Asn Ala Gln Ala Gln Gln

120 125

DNA sequence #A10 pMON13252 50D

ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA

[SEQ ID NO:216]

GCCACCGCTG CCGCTGCTGG ACTTCAACAA CCTCAATGGT GAAGACCAAG

ATATCCTGAT GGAAAATAAC CTTCGTCGTC CAAACCTCGA GGCATTCAAC

CGTGCTGTCA ACTCTCTGCA GAATGCATCA GCAATTGAGA GCATTCTTAA

AAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC ACGCGACATC

CAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACC

TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAG

TABLE 7

position/

mutant

NATIVE

SUBSTITUTION

RELATIVE

pMON number

(15-125) hiL-3

amino acid

amino acid

SEQ ID NO:

POTENCY

pMON13201

45/31M

Gln

Met

19

6.3

pMON13202

51/37R

Asn

Arg

19

1.58

pMON13203

51/37P

Asn

Pro

19

2.5

pMON13204

51/37T

Asn

Thr

19

3.16

pMON13205

56/42S

Pro

Ser

19

6.3

pMON13206

98/84I

His

Ile

19

6.3

pMON13207

45/31V

Gln

Val

19

4

pMON13208

42/28D

Gly

Asp

19

6.3

pMON13209

42/28S

Gly

Ser

19

12.6

pMON13210

42/28A

Gly

Ala

19

2.5

pMON13211

46/32S

Asp

Ser

19

16

pMON13212

82/68W

Leu

Trp

19

5

pMON13213

82/68D

Leu

Asp

19

4

pMON13214

100/86R

Lys

Arg

19

4

pMON13215

116/102W

Lys

Trp

19

31

pMON13216

23/9L

Ile

Leu

19

4

pMON13217

32/18R

Leu

Arg

19

7.9

pMON13218

32/18N

Leu

Asn

19

2

pMON13219

32/18A

Leu

Ala

19

1.58

pMON13220

34/20S

Leu

Ser

19

6.3

pMON13221

34/20M

Leu

Met

19

6.3

pMON13222

50/36D

Glu

Asp

19

7.9

pMON13223

62/48I

Asn

Ile

19

*

pMON13224

166/52R

Lys

Arg

19

4

pMON13225

176/62P

Ser

Pro

19

1.25

pMON13226

77/63L

Ile

Leu

19

1.58

pMON13227

22/8G

Glu

Gly

19

0.008

pMON13228

115/101M

Leu

Met

19

0.04

pMON13229

122/108I

Gln

Ile

19

1

pMON13231

51/37H

Asn

His

19

1.25

pMON13232

59/45L

Glu

Leu

19

1.99

pMON13233

63/49H

Arg

His

19

*

pMON13234

64/50N

Ala

Asn

19

0.03

pMON13235

65/51T

Val

Thr

19

1.58

pMON13236

76/62V

Ser

Val

19

2.5

pMON13237

76/62A

Ser

Ala

19

5

pMON13238

91/77P

Ala

Pro

19

*

pMON13240

100/86Q

Lys

Gln

19

2.5

pMON13241

101/87M

Asp

Met

19

6.3

pMON13242

105/91N

Asn

Asn

19

*

pMON13243

116/102V

Lys

Val

19

79

pMON13244

122/108F

Gln

Phe

19

6.3

pMON13245

123/109E

Ala

Gln

19

1.58

position/

mutant

NATIVE

SUBSTITUTION

RELATVE

pMON number

(1-133) hiL-3

amino acid

amino acid

SEQ ID NO:

POTENCY

pMON13246

42D

Gly

Asp

15

20

pMON13247

42S

Gly

Ser

15

*

pMON13248

42A

Gly

Ala

15

16

pMON13249

45V

Gln

Val

15

5

pMON13250

45M

Gln

Met

15

*

pMON13251

46S

Asp

Ser

15

5

pMON13252

50D

Glu

Asp

15

5

pMON13253

981

His

Ile

15

*

pMON13264

97V

Ile

Val

15

4

pMON13266

75K

Glu

Lys

15

0.25

pMON13267

89N

Thr

Asn

15

2.5

Table 7 shows the biological activity of (15-125) hIL-3 mutant polypeptides of the present invention expressed from Intracellular expression vectors. Upon expression these muteins may have Met- or Met-Ala-preceding the initial (15-125) hIL-3 amino acid. The relative biological activity of IL-3 mutants is calculated by dividing the EC

50

(1-133) hIL-3 by the EC

50

of the mutant.

EXAMPLE 66

The variants in Table 8 were constructed by cassette mutagenesis using methods described in the Materials and Methods and the Examples contained herein, particularly Examples 54-57. Parental plasmid DNA (Table 8), digested with the appropriate restriction enzymes (Table 8), was ligated with the indicated annealed pairs of complementary oligonucleotides (Table 8). The assembled oligonucleotides create appropriate restriction ends and a portion of the (15-125) hIL-3 gene sequence Individual isolates were screened by restriction analysis and DNA sequenced to confirm that the desired changes in the (15-125) hIL-3 variant gene were made. The oligonucleotides create change(s) in the (15-125) hIL-3 gene which enclode the corresponding amino acid substitution in the variant polypeptide (Table 8). The amino acids substitutions in polypeptide #1 (SEQ ID NO:65) are indicated in Table 8.

position/

sub-

restriction

native a.a.

stitution

codon

oligo pair

oligo pair

oligo pair

oligo pair

oligo pair

parental plasmid

digest

21 asp

glu

GAA

21glu1

NcoRv2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:73

SEQ ID NO:523

SEQ ID NO:524

21glu4

NcoRV5

NcoRV6

SEQ ID NO:219

SEQ ID NO:526

SEQ ID NO:527

21 asp

gln

CAA

21gln1

NcoRv2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:71

SEQ ID NO:523

SEQ ID NO:524

21gln4

NcoRV5

NcoRV6

SEQ ID NO:72

SEQ ID NO:526

SEQ ID NO:527

21 asp

asn

AAC

21asn1

NcoRv2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:68

SEQ ID NO:523

SEQ ID NO:524

21asn4

NcoRV5

NcoRV6

SEQ ID NO:70

SEQ ID NO:526

SEQ ID NO:527

21 asp

thr

ACC

21thr1

NcoRv2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:232

SEQ ID NO:523

SEQ ID NO:524

21thr4

NcoRV5

NcoRV6

SEQ ID NO:233

SEQ ID NO:526

SEQ ID NO:527

21 asp

ser

AGC

21ser1

NcoRv2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:230

SEQ ID NO:523

SEQ ID NO:524

21ser4

NcoRV5

NcoRV6

SEQ ID NO:231

SEQ ID NO:526

SEQ ID NO:527

22 glu

asp

GAC

22asp1

NcoRv2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:236

SEQ ID NO:523

SEQ ID NO:524

22asp4

NcoRV5

NcoRV6

SEQ ID NO:237

SEQ ID NO:526

SEQ ID NO:527

22 glu

asn

AAC

22asn1

NcoRv2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:234

SEQ ID NO:523

SEQ ID NO:524

22asn4

NcoRV5

NcoRV6

SEQ ID NO:235

SEQ ID NO:526

SEQ ID NO:527

22 glu

gln

CAG

22gln1

NcoRv2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:238

SEQ ID NO:523

SEQ ID NO:524

22gln4

NcoRV5

NcoRV6

SEQ ID NO:239

SEQ ID NO:526

SEQ ID NO:521

22 glu

leu

CTG

22leu1

NcoRv2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:240

SEQ ID NO:523

SEQ ID NO:524

22leu4

NcoRV5

NcoRV6

SEQ ID NO:241

SEQ ID NO:526

SEQ ID NO:527

22 glu

val

GTT

22val1

NcoRv2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:242

SEQ ID NO:523

SEQ ID NO:524

22val4

NcoRV5

NcoRV6

SEQ ID NO:243

SEQ ID NO:526

SEQ ID NO:527

34 leu

glu

GAA

NcoRV1

34Glu2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:251

SEQ ID NO:524

NcoRV4

34Glu5

NcoRV6

SEQ ID NO:525

SEQ ID NO:252

SEQ ID NO:527

34 leu

gln

GAG

NcoRV1

34gln2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:248

SEQ ID NO:524

NcoRV4

34gln5

NcoRV6

SEQ ID NO:525

SEQ ID NO:249

SEQ ID NO:527

34 leu

thr

ACC

NcoRV1

34thr2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:256

SEQ ID NO:524

NcoRV4

34thr5

NcoRV6

SEQ ID NO:525

SEQ ID NO:257

SEQ ID NO:527

34 leu

arg

CGT

NcoRV1

34arg2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:246

SEQ ID NO:524

NcoRV4

34arg5

NcoRV6

SEQ ID NO:525

SEQ ID NO:247

SEQ ID NO:527

34 leu

ala

GCT

NcoRV1

34ala2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:244

SEQ ID NO:524

NcoRV4

34ala5

NcoRV6

SEQ ID NO:525

SEQ ID NO:245

SEQ ID NO:527

34 leu

phe

TTC

NcoRV1

34phe2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:254

SEQ ID NO:524

NcoRV4

34phe5

NcoRV6

SEQ ID NO:525

SEQ ID NO:255

SEQ ID NO:527

34 leu

ile

ATC

NcoRV1

34ile2

NcoRV3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:252

SEQ ID NO:524

NcoRV4

34ile5

NcoRV6

SEQ ID NO:525

SEQ ID NO:253

SEQ ID NO:527

42 gly

lys

AAA

NcoRV1

NcoRv2

42lys3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:268

NcoRV4

NcoRV5

42lys6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:269

42 gly

asn

AAC

NcoRV1

NcoRv2

42asn3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:260

NcoRV4

NcoRV5

42asn6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:261

42 gly

thr

ACC

NcoRV1

NcoRv2

42thr3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:274

NcoRV4

NcoRV5

42thr6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:275

42 gly

leu

CTG

NcoRV1

NcoRv2

42leu3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:266

NcoRV4

NcoRV5

42leu6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:267

42 gly

val

GTT

NcoRV1

NcoRv2

42val3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:278

NcoRV4

NcoRV5

42val6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:279

42 gly

glu

GAA

NcoRV1

NcoRv2

42glu3

PMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:262

NcoRV4

NcoRV5

42glu6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:263

42 gly

phe

TTC

NcoRV1

NcoRv2

42phe3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:272

NcoRV4

NcoRV5

42phe6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:273

42 gly

tyr

TAC

NcoRV1

NcoRv2

42tyr3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:276

NcoRV4

NcoRV5

42tyr6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:277

42 gly

ile

ATC

NcoRV1

NcoRv2

42ile3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:264

NcoRV4

NcoRV5

42ile6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:265

42 gly

met

ATG

NcoRV1

NcoRv2

42met3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:270

NcoRV4

NcoRV5

42met6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:271

43 glu

gln

CAG

NcoRV1

NcoRv2

43gln3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:282

NcoRV4

NcoRV5

43gln6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:283

43 glu

arg

CGT

NcoRV1

NcoRv2

43arg3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:280

NcoRV4

NcoRV5

43arg6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:281

43 glu

thr

ACC

NcoRV1

NcoRv2

43thr3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:286

NcoRV4

NcoRV5

43thr6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:287

43 glu

gly

GGT

NcoRV1

NcoRv2

43gly3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:284

NcoRV4

NcoRV5

43gly6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:285

44 asp

glu

GAA

NcoRV1

NcoRv2

44glu3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:294

NcoRV4

NcoRV5

44glu6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:295

44 asp

asn

AAC

NcoRV1

NcoRv2

44asn3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:290

NcoRV4

NcoRV5

44asn6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:291

44 asp

gln

CAG

NcoRV1

NcoRv2

44gln3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:292

NcoRV4

NcoRV5

44gln6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:293

44 asp

ala

GCT

NcoRV1

NcoRv2

44ala3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:288

NcoRV4

NcoRV5

44ala6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:289

45 gln

asp

GAC

NcoRV1

NcoRv2

45asp3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:302

NcoRV4

NcoRV5

45asp6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:303

45 gln

asn

AAC

NcoRV1

NcoRv2

45asn3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:300

NcoRV4

NcoRV5

45asn6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:301

45 gln

arg

CGT

NcoRV1

NcoRv2

45arg3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:298

NcoRV4

NcoRV5

45arg6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:299

45 gln

ser

TCC

NcoRV1

NcoRv2

45ser3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:310

NcoRV4

NcoRV5

45ser6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:311

45 gln

ala

GCT

NcoRV1

NcoRv2

45ala3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:296

NcoRV4

NcoRV5

45ala6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:297

45 gln

ile

ATC

NcoRV1

NcoRv2

45ile3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:308

NcoRV4

NcoRV5

45ile6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:309

45 gln

glu

GAA

NcoRV1

NcoRv2

45glu3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:304

NcoRV4

NcoRV5

45glu6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:305

45 gln

his

CAC

NcoRV1

NcoRv2

45his3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:306

NcoRV4

NcoRV5

45his6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:307

46 asp

glu

GAA

NcoRV1

NcoRv2

46glu3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:318

NcoRV4

NcoRV5

46glu6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:319

46 asp

asn

AAC

NcoRV1

NcoRv2

46asn3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:314

NcoRV4

NcoRV5

46asn6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:315

46 asp

gln

CAG

NcoRV1

NcoRv2

46gln3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:316

NcoRV4

NcoRV5

46gln6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:317

46 asp

lys

AAA

NcoRV1

NcoRv2

46lys3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:326

NcoRV4

NcoRV5

46lys6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:327

46 asp

his

CAC

NcoRV1

NcoRv2

48his3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:320

NcoRV4

NcoRV5

46his6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:321

46 asp

ala

GCT

NcoRV1

NcoRv2

48ala3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:312

NCORV4

NcoRV5

48ala6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:313

46 asp

tyr

TAC

NcoRV1

NcoRv2

46tyr3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:328

NcoRV4

NcoRV5

46tyr6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:329

46 asp

ile

ATC

NcoRV1

NcoRv2

46ile3

pMON13356

Ncol, EcoRV

SEQ ID NO: 522

SEQ ID NO:523

SEQ ID NO:322

NcoRV4

NcoRV5

46ile6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:323

46 asp

val

GTT

NcoRV1

NcoRV2

46val3

pMON13356

Ncol, EcoRV

SEQ ID NO:522

SEQ ID NO:523

SEQ ID NO:330

NcoRV4

NcoRV5

46val6

SEQ ID NO:525

SEQ ID NO:526

SEQ ID NO:331

48 leu

glu

GAA

48glu1

RVNsl2

RVNsl3

pMON13367

EcoRV, Ncol

SEQ ID NO:334

SEQ ID NO:528

SEQ ID NO:530

48glu4

RVNsl5

RVNsl6

SEQ ID NO:535

SEQ ID NO:532

SEQ ID NO:533

48 leu

lys

AAA

48lys1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:336

SEQ ID NO:529

SEQ ID NO:530

48lys4

RVNsl5

RVNSl6

SEQ ID NO:337

SEQ ID NO:532

SEQ ID NO:529

48 leu

thr

ACC

48thr1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:340

SEQ ID NO:525

SEQ ID NO:530

48thr4

RVNsl5

RVNsl5

SEQ ID NO:341

SEQ ID NO:532

SEQ ID NO:533

48 leu

ala

GCT

48ala1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:332

SEQ ID NO:529

SEQ ID NO:530

48ala4

RVNsl5

RVNsl6

SEQ ID NO:333

SEQ ID NO:532

SEQ ID NO:533

48 leu

met

ATG

48met1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:338

SEQ ID NO:529

SEQ ID NO:530

48met4

RVNsl5

RVNsl6

SEQ ID NO:339

SEQ ID NO:532

SEQ ID NO:533

48 leu

val

CAC

48val1

RVNsl2

RVNs13

pMON13357

EcoRV, Ncol

SEQ ID NO:342

SEQ ID NO:529

SEQ ID NO:530

48val4

RVNsl5

RVNsl6

SEQ ID NO:343

SEQ ID NO:532

SEQ ID NO:533

50 glu

lys

AAA

50lys1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:356

SEQ ID NO:529

SEQ ID NO:530

501ys4

RVNsl5

RVNsl5

SEQ ID NO:357

SEQ ID NO:532

SEQ ID NO:533

50 glu

asn

AAC

50asn1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:352

SEQ ID NO:529

SEQ ID NO:530

50asn4

RVNsl5

RVNsl6

SEQ ID NO:353

SEQ ID NO:532

SEQ ID NO:533

50 glu

ser

TCC

50ser1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:358

SEQ ID NO:529

SEQ ID NO:530

50ser4

RVNsl5

RVNsl6

SEQ ID NO:359

SEQ ID NO:532

SEQ ID NO:533

50 glu

ala

GCT

50ala1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:350

SEQ ID NO:529

SEQ ID NO:530

50ala4

RVNsl5

RVNsl6

SEQ ID NO:351

SEQ ID NO:532

SEQ ID NO:533

50 glu

ile

ATC

50ile1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:354

SEQ ID NO:529

SEQ ID NO:530

50ile4

RVNsl5

RVNsl6

SEQ ID NO:355

SEQ ID NO:532

SEQ ID NO:533

50 glu

val

GTT

50val1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:360

SEQ ID NO:529

SEQ ID NO:530

50val4

RVNsl5

RVNsl6

SEQ ID NO:361

SEQ ID NO:532

SEQ ID NO:533

50 glu

his

CAC

50his1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:344

SEQ ID NO:529

SEQ ID NO:530

50his4

RVNsl5

RVNsl6

SEQ ID NO:345

SEQ ID NO:532

SEQ ID NO:533

50 glu

phe

TTC

50phe1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:348

SEQ ID NO:529

SEQ ID NO:530

50phe4

RVNsl5

RVNsl6

SEQ ID NO:349

SEQ ID NO:532

SEQ ID NO:533

50 glu

met

ATG

50met1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:346

SEQ ID NO:529

SEQ ID NO:530

50met4

RVNsl5

RVNsl6

SEQ ID NO:347

SEQ ID NO:532

SEQ ID NO:533

54 arg

asn

AAC

54asn1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:364

SEQ ID NO:529

SEQ ID NO:530

54asn4

RVNsl5

RVNsl6

SEQ ID NO:365

SEQ ID NO:532

SEQ ID NO:533

54 arg

lys

AAA

54lys1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:368

SEQ ID NO:529

SEQ ID NO:530

54lys4

RVNsl5

RVNsl6

SEQ ID NO:369

SEQ ID NO:532

SEQ ID NO:533

54 arg

his

CAC

54his1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:366

SEQ ID NO:529

SEQ ID NO:53O

54his4

RVNsl5

RVNsl6

SEQ ID NO:367

SEQ ID NO:532

SEQ ID NO:533

54 arg

ala

GCT

54ala1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:362

SEQ ID NO:529

SEQ ID NO:530

54ala4

RVNsl5

RVNsl6

SEQ ID NO:363

SEQ ID NO:532

SEQ ID NO:533

56 pro

glu

GAA

56glu1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:376

SEQ ID NO:529

SEQ ID NO:530

RVNsl4

56glu5

RVNsl6

SEQ ID NO:531

SEQ ID NO:377

SEQ ID NO:533

56 pro

gln

56gln1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:314

SEQ ID NO:529

SEQ ID NO:530

RVNsl4

56gln5

RVNsl6

SEQ ID NO:531

SEQ ID NO:375

SEQ ID NO:533

58 pro

arg

CGT

56arg1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:372

SEQ ID NO:529

SEQ ID NO:530

RVNsl4

56arg5

RVNsl6

SEQ ID NO:531

SEQ ID NO:373

SEQ ID NO:533

56 pro

his

CAC

56his1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:378

SEQ ID NO:529

SEQ ID NO:530

RVNsl4

56his5

RVNsl6

SEQ ID NO:531

SEQ ID NO:379

SEQ ID NO:533

56 pro

thr

ACC

56thr1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:384

SEQ ID NO:529

SEQ ID NO:530

RVNsl4

56thr5

RVNsl6

SEQ ID NO:531

SEQ ID NO:385

SEQ ID NO:533

56 pro

ala

GCT

56ala1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:370

SEQ ID NO:529

SEQ ID NO:530

RVNsl4

56ala5

RVNsl6

SEQ ID NO:531

SEQ ID NO:371

SEQ ID NO:533

56 pro

tyr

TAC

56tyr1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:386

SEQ ID NO:529

SEQ ID NO:530

RVNsl4

56tyr5

RVNsl6

SEQ ID NO:531

SEQ ID NO:387

SEQ ID NO:533

56 pro

phe

TTC

56phe1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:382

SEQ ID NO:529

SEQ ID NO:530

RVNsl4

56phe5

RVNsl6

SEQ ID NO:531

SEQ ID NO:383

SEQ ID NO:533

56 pro

leu

CTG

56leu1

RVNsl2

RVNsl3

pMON133S1

EcoRV, Ncol

SEQ ID NO:380

SEQ ID NO:529

SEQ ID NO:530

RVNsl4

56leu5

RVNsl6

SEQ ID NO:531

SEQ ID NO:381

SEQ ID NO:533

56 pro

val

GTT

56val1

RVNsl2

RVNsl3

pMON13357

EcoRV, Ncol

SEQ ID NO:388

SEQ ID NO:529

SEQ ID NO:530

RVNsl4

56val5

RVNsl6

SEQ ID NO:531

SEQ ID NO:389

SEQ ID NO:533

82 leu

glu

GAA

NslEco1

82glu2

NslEco3

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:94

SEQ ID NO:536

SEQ ID NO:539

NslEco5

82glu6

NslEco7

NslEco8

SEQ ID NO:540

SEQ ID NO:395

SEQ ID NO:542

SEQ ID NO:545

82 leu

asn

AAC

NslEcol

82asn2

NslEco3

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:392

SEQ ID NO:536

SEQ ID NO:539

NslEco5

82asn6

NslEco7

NslEco8

SEQ ID NO:540

SEQ ID NO:393

SEQ ID NO:542

SEQ ID NO:545

82 leu

his

CAC

NslEco1

82his2

NslEco3

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:396

SEQ ID NO:538

SEQ ID NO:539

NslEco5

82his6

NslEco7

NslEco8

SEQ ID NO:540

SEQ ID NO:397

SEQ ID NO:542

SEQ ID NO:545

82 leu

thr

ACC

NslEco1

82thr2

NslEco3

NslEc04

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:406

SEQ ID NO:536

SEQ ID NO:539

NslEco5

82thr6

NslEco7

NslEco3

SEQ ID NO:540

SEQ ID NO:407

SEQ ID NO:542

SEQ ID NO:545

82 leu

ser

TCC

NslEco1

82ser2

NslEco3

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:404

SEQ ID NO:538

SEQ ID NO:539

NslEco5

82ser6

NslEco7

NslEco8

SEQ ID NO:540

SEQ ID NO:405

SEQ ID NO:542

SEQ ID NO:545

82 leu

ala

GCT

NslEco1

82ala2

NslEco3

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:390

SEQ ID NO:536

SEQ ID NO:539

NslEco5

82ala6

NslEco7

NslEco8

SEQ ID NO:540

SEQ ID NO:391

SEQ ID NO:542

SEQ ID NO:545

82 leu

tyr

TAC

NslEco1

82tyr2

NslEco3

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:408

SEQ ID NO:536

SEQ ID NO:539

NslEco5

82tyr6

NslEco7

NslEco8

SEQ ID NO:540

SEQ ID NO:409

SEQ ID NO:542

SEQ ID NO:545

82 leu

phe

TTC

NslEco1

82phe2

NslEco3

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:402

SEQ ID NO:536

SEQ ID NO:539

NslEco5

82phe6

NslEco7

NslEco8

SEQ ID NO:540

SEQ ID NO:403

SEQ ID NO:542

SEQ ID NO:545

82 leu

ile

ATC

NslEco1

82ile2

NslEco3

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:398

SEQ ID NO:536

SEQ ID NO:539

NslEco5

82ile6

NslEco7

NslEco8

SEQ ID NO:540

SEQ ID NO:399

SEQ ID NO:542

SEQ ID NO:545

82 leu

met

ATG

NslEco1

82met2

NslEco3

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:400

SEQ ID NO:536

SEQ ID NO:539

NslEco5

82met6

NslEco7

NslEco8

SEQ ID NO:540

SEQ ID NO:401

SEQ ID NO:542

SEQ ID NO:545

92 pro

ala

GCT

NslEco1

NslEco2

92ala3A

NslEco3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:410

SEQ ID NO:538

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

92ala7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:411

SEQ ID NO:545

92 pro

gly

GGT

NslEco1

NslEco2

92gly3A

NslEco3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:412

SEQ ID No:538

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

92ala7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:413

SEQ ID NO:545

92 pro

ile

ATC

NslEco1

NslEco2

92ile3A

NslEco3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:414

SEQ ID NO:538

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

92ala7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:415

SEQ ID NO:545

94 arg

gln

GAG

NslEco1

NslEco2

NslEco3A

94gln3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:418

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

94gln7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:419

SEQ ID NO:545

94 arg

lys

AAA

NslEco1

NslEco2

NslEco3A

94lys3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:422

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

94lys7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:423

SEQ ID NO:545

94 arg

his

CAC

NslEco1

NslEco2

NslEco3A

94his3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:420

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

94his7B

NslEco8

SEQ ID NO:540

SEQ ID NO:538

SEQ ID NO:543

SEQ ID NO:421

SEQ ID NO:545

94 arg

ala

GCT

NslEco1

NslEco2

NslEco3A

94ala3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:416

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

94ala7B

NslEco8

SEQ ED NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:417

SEQ ID NO:545

95 his

asn

AAC

NslEco1

NslEco2

NslEco3A

95asn3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:426

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

95asn7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:427

SEQ ID NO:545

95 his

lys

AAA

NslEco1

NslEco2

NslEco3A

95lys3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:432

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

95lys7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:433

SEQ ID NO:545

95 his

ser

TCC

NslEco1

NslEco2

NslEco3A

95ser3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:438

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

95ser7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:439

SEQ ID NO:545

95 his

ala

GCT

NslEco1

NslEco2

NslEco3A

95ala3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:424

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

95ala7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:425

SEQ ID NO:545

95 his

trp

TGG

NslEco1

NslEco2

NslEco3A

95trp3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:440

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

95trp7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:441

SEQ ID NO:545

95 his

phe

TTC

NslEco1

NslEco2

NslEco3A

95phe3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO11

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

95phe7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:437

SEQ ID NO:545

95 his

ile

ATC

NslEco1

NslEco2

NslEco3A

95ile3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:430

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

95ile7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID No:431

SEQ ID NO:545

98 his

glu

GAA

NslEco1

NslEco2

NslEco3A

98glu3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID No:446

SEQ ID NO:539

NslEco5

NslEco5

NslEco7A

98glu7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID No:447

SEQ ID NO:545

98 his

gln

CAA

NslEco1

NslEco2

NslEco3A

98gln3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID No:444

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

98gln7B

NslEco8

SEQ ID NQ:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:445

SEQ ID NO:545

98 his

ser

TCC

NslEco1

NslEco2

NslEco3A

98ser3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID No:452

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

98ser7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID No:453

SEQ ID NO:545

98 his

phe

TTC

NslEco1

NslEco2

NslEco3A

98phe3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:450

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

98phe7B

NslEco3

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:451

SEQ ID NO:545

98 his

met

ATG

NslEco1

NslEco2

NslEco3A

98met3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:446

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

98met7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:449

SEQ ID NO:545

98 his

val

GTA

NslEco1

NslEco2

NslEco3A

98val3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:454

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

98val7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:455

SEQ ID NO:545

98 his

lys

AAA

NslEco1

NslEco2

NslEco3A

98lys3B

NsilEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:458

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

98lys7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:459

SEQ ID NO:545

98 his

arg

CGT

NslEco1

NslEco2

NslEco3A

98arg3B

NslEco4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:456

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

98arg7B

NslEco8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:457

SEQ ID NO:545

98 his

tyr

TAC

NslEco1

NslEco2

NslEco3A

98tyr3B

NslEc04

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:537

SEQ ID NO:460

SEQ ID NO:539

NslEco5

NslEco6

NslEco7A

98tyr7B

NslEco5

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:543

SEQ ID NO:461

SEQ ID NO:545

101 asp

glu

GAA

NslEco1

NslEco2

NslEco3

101glu4

pMON13358

Nsil, EcoRI

SEQ ED NO:534

SEQ ID NO:535

SEQ ID NO:536

SEQ ID NO:466

NslEco5

NslEco6

NslEco7

101glu8

SEQ ED NO:540

SEQ ID NO:541

SEQ ID NO:542

SEQ ID NO:467

101 asp

asn

AAC

NslEco1

NslEco2

NslEco3

101asn4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:536

SEQ ID NO:464

NslEco5

NslEco6

NslEco7

101asn8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:542

SEQ ID NO:465

101 asp

ser

TTC

NslEco1

NslEco2

NslEco3

101ser4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:536

SEQ ID NO:476

NslEco5

NslEco6

NslEco7

101ser8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:542

SEQ ID NO:477

101 asp

ala

GCT

NslEco1

NslEco2

NslEco3

101ala4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:536

SEQ ID NO:462

NslEco5

NslEco6

NslEco7

101ala8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:542

SEQ ID NO:463

101 asp

gly

GGT

NslEco1

NslEco2

NslEco3

101gly4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:335

SEQ ID NO:536

SEQ ID NO:466

NslEco5

NslEco6

NslEco7

101gly8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:542

SEQ ID NO:469

101 asp

ile

ATC

NslEco1

NslEco2

NslEco3

101ile4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:536

SEQ ID NO:470

NslEco5

NslEco6

NslEco7

101ile8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:542

SEQ ID NO:471

101 asp

leu

CTG

NslEco1

NslEco2

NslEco3

101leu4

pMON13358

Nsil, EcoRI

SEQ ID NO:534

SEQ ID NO:535

SEQ ID NO:536

SEQ ID NO:472

NslEco5

NslEco6

NslEco7

101leu8

SEQ ID NO:540

SEQ ID NO:541

SEQ ID NO:542

SEQ ID NO:473

108 arg

gln

CAG

108gln1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:480

SEQ ID NO:547

108gln3

EcoHin4

SEQ ID NO:481

SEQ ID NO:549

108 arg

his

CAC

108his1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:482

SEQ ID NO:547

108his3

EcoHin4

SEQ ID NO:483

SEQ ID NO:549

108 arg

ser

TCC

108ser1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:484

SEQ ID NO:547

108ser3

EcoHin4

SEQ ID NO:485

SEQ ID NO:549

108 arg

ala

GCT

108ala1

EcoHin2

pMON13359

EcoRi, HinDIII

SEQ ID NO:478

SEQ ID NO:547

108ala3

EcoHin4

SEQ ID NO:479

SEQ ID NO:549

110 lys

arg

CGT

110arg1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:46

SEQ ID NO:547

110arg3

EcoHin4

SEQ ID NO:487

SEQ ID NO:549

110 lys

his

CAC

110his1

EcoHin2

pMON13359

EcoRI, HinDILL

SEQ ID NO:490

SEQ ID NO:547

110his3

EcoHin4

SEQ ID NO:491

SEQ ID NO:549

110 lys

glu

GAA

110glu1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:488

SEQ ID NO:547

110glu3

EcoHin4

SEQ ID NO:499

SEQ ID NO:549

110 lys

ser

TCC

110ser1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:494

SEQ ID NO:547

110ser3

EcoHin4

SEQ ID NO:495

SEQ ID NO:549

110 lys

ala

GCT

110ala1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:492

SEQ ID NO:547

110la3

EcoHin4

SEQ ID NO:493

SEQ ID NO:549

113 phe

asp

GAC

113asp1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:496

SEQ ID NO:547

113asp3

EcoHin4

SEQ ID NO:497

SEQ ID NO:549

113 phe

lys

AAA

113lys1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:502

SEQ ID NO:547

113lys3

EcoHin4

SEQ ID NO:503

SEQ ID NO:549

113 phe

leu

CTG

113leu1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:500

SEQ ID NO:547

113leu3

EcoHin4

SEQ ID NO:501

SEQ ID NO:549

113 phe

ile

ATC

113ile1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:498

SEQ ID NO:541

113ile3

EcoHin4

SEQ ID NO:499

SEQ ID NO:549

113 phe

val

GTT

113val1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:504

SEQ ID NO:547

113va13

EcoHin4

SEQ ID NO:505

SEQ ID NO:549

116 lys

asn

AAC

116asn1

EcoHin2

pMONI3359

EcoRI, HinDIII

SEQ ID NO:510

SEQ ID NO:547

116asn3

EcoHin4

SEQ ID NO:511

SEQ ID NO:549

116 lys

arg

CGT

116arg1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:506

SEQ ID NO:547

116arg3

EcoHin4

SEQ ID NO:509

SEQ ID NO:549

116 lys

his

CAC

116his1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:514

SEQ ID NO:547

116his3

EcoHin4

SEQ ID NO:515

SEQ ID NO:549

116 lys

ala

GCT

116ala1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:506

SEQ ID NO:547

116ala3

EcoHin4

SEQ ID NO:507

SEQ ID NO:549

116 lys

tyr

TAC

116tyr1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:520

SEQ ID NO:547

116tyr3

EcoHin4

SEQ ID NO:521

SEQ ID NO:549

116 lys

phe

TTC

116phe1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:518

SEQ ID NO:547

116phe3

EcoHin4

SEQ ID NO:519

SEQ ID NO:549

116 lys

gln

CAG

116gln1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:512

SEQ ID NO:547

116gln3

EcoHin4

SEQ ID NO:513

SEQ ID NO:549

116 lys

met

ATG

116met1

EcoHin2

pMON13359

EcoRI, HinDIII

SEQ ID NO:516

SEQ ID NO:547

116met3

EcoHin4

SEQ ID NO:517

SEQ ID NO:549

It will be apparent to those skilled in the art that other codons besides those shown in Table 8 can also code for the substituted amino acids in the hIL-3 muteins. The present invention includes the DNAs encoding the mutant hIL-3 polypeptides of the invention including the various codons which can code for the parental and substituted amino acids of the hIL-3 muteins of the invention due to the degeneracy of the genetic code.

hIL-3 (15-125) variant genes encoding the variants listed in Table 8 can also be expressed from intracellular expression vectors to produce large quantities of the variant protein which can be purified and assayed for biological activity. The hIL-3 variant genes, from Table 8, can be excised from the secretion expression vector, as a 345 base pair NcoI/HindIII fragment and ligated into an appropriate intrecellular expression vector, such as pMON2341 digested with NcoI and HindIII.

Table 9 shows the biological activity of (15-125) hIL-3 muteins of the present invention which have one amino acid substitutions in the (15-125) hIL-3 polypeptide and which were constructed as described in Example 66. The mutants in Table 9 were secreted into the periplasmic space in

E. coli.

The periplasmic content was released by osmotic shock and the material in the crude osmotic shock fraction was screened for growth promoting activity. Biological activity is the growth promoting activity of AML cells relative to (1-125) hIL-3 (pMON6458 or pMMON5988). The relative biological activity of IL-3 mutants is calculated by dividing the EC

50

(1-133) hIL-3 by the EC

50

of the mutant. The numbers in parentheses indicate the number of repeat assays. When a variant was assayed more than once the standard deviation is indicated. An “*”

0

indicates that the hIL3 variant protein level was less than 1.0 μg/ml and was not screened for growth promoting activity.

TABLE 9

(15-125) hIL-3 MUTANT

PARENTAL

BIOL

aa position

AA

codon

AA

SEQ ID NO:

codon

ACTIVITY

21/7

ASP

GAT

ASN

19

AAC

0.01

21/7

ASP

GAT

GLN

19

CAA

0.07

21/7

ASP

GAT

GLU

19

GAA

0.5

21/7

ASP

GAT

SER

19

AGC

0.1

21/7

ASP

GAT

THR

19

ACC

0.1

22/8

GLU

GAA

ASN

19

AAC

*

22/8

GLU

GAA

ASP

19

GAC

*

22/8

GLU

GAA

GLN

19

CAG

<0.01

22/8

GLU

GAA

LEU

19

CTG

*

22/8

GLU

GAA

VAL

19

GTT

*

34/20

LEU

TTG

ALA

19

GCT

2.2

34/20

LEU

TTG

ARG

19

CGT

2.2

34/20

LEU

TTG

GLN

19

CAG

1.1

34/20

LEU

TTG

GLU

19

GAA

1.5

34/20

LEU

TTG

ILE

19

ATC

1.3

34/20

LEU

TTG

PHE

19

TTC

1.8

34/20

LEU

TTG

THR

19

ACC

1.1

42/28

GLY

GGG

ASN

19

AAC

1.3 (3) 0.28

42/28

GLY

GGG

ILE

19

ATC

10

42/28

GLY

GGG

LEU

19

CTG

10.1 (3) 7.57

42/28

GLY

GGG

MET

19

ATG

2.2 (3) 1.14

42/28

GLY

GGG

TYR

19

TAC

11 (2) 8.9

42/28

GLY

GGG

VAL

19

GTT

0.33

43/29

GLU

GAA

ARG

19

CGT

*

43/29

GLU

GAA

GLN

19

CAG

<0.004

43/29

GLU

GAA

GLY

19

GGT

*

43/29

GLU

GAA

THR

19

ACC

0.005

44/30

ASP

GAC

ALA

19

GCT

*

44/30

ASP

GAC

ASN

19

AAC

*

44/30

ASP

GAC

GLN

19

CAG

*

44/30

ASP

GAC

GLU

19

GAA

0.66

45/31

GLN

CAA

ALA

19

GCT

1

45/31

GLN

CAA

ASN

19

AAC

15.8

45/31

GLN

CAA

GLU

19

GAA

2.3

45/31

GLN

CAA

ILE

19

ATC

4.9

45/31

GLN

CAA

SER

19

TCC

0.7

46/32

ASP

GAC

ALA

19

GCT

6.3

46/32

ASP

GAC

ASN

19

AAC

0.66, 1.1

46/32

ASP

GAC

GLN

19

CAG

6.3

46/32

ASP

GAC

GLU

19

GAA

1.97 (3) 2.14

46/32

ASP

GAC

HIS

19

CAC

3.2, 1.4

46/32

ASP

GAC

ILE

19

ATC

0.5

46/32

ASP

GAC

LYS

19

AAA

0.5

46/32

ASP

GAC

TYR

19

TAC

0.66

46/32

ASP

GAC

VAL

19

GTT

6.3

48/34

LEU

CTG

GLU

19

GAA

*

48/34

LEU

CTG

HIS

19

*

48/34

LEU

CTG

LYS

19

AAA

*

48/34

LEU

CTG

THR

19

ACC

*

48/34

LEU

CTG

VAL

19

CAC

*

50/36

GLU

GAA

ALA

19

GCT

0.5

50/36

GLU

GAA

ASN

19

AAC

1.7

50/36

GLU

GAA

HIS

19

CAC

*

50/36

GLU

GAA

LYS

19

AAA

*

50/36

GLU

GAA

SER

19

TCC

1.3

50/36

GLU

GAA

VAL

19

GTT

*

54/40

ARG

CGA

ALA

19

GCT

0.9

54/40

AFG

CGA

ASN

19

AAC

*

54/40

ARG

CGA

HIS

19

CAC

0.01

54/40

ARG

CGA

LYS

19

AAA

0.2

56/42

PRO

CAA

ALA

19

GCT

1.8

56/42

PRO

CAA

ASN

19

0.6

56/42

PRO

CAA

ARG

19

CGT

1.2

56/42

PRO

CAA

GLU

19

GAA

0.9

56/42

PRO

CAA

HIS

19

CAC

0.4

56/42

PRO

CAA

LEU

19

CTG

1.2

56/42

PRO

CAA

PHE

19

TTC

56/42

PRO

CAA

THR

19

ACC

0.6

56/42

PRO

CAA

VAL

19

GTT

1.1

82/68

LEU

CTG

ALA

19

GCT

0.5

82/68

LEU

CTG

ASN

19

AAC

2.9

82/68

LEU

CTG

GLU

19

GAA

4.57 (3) 5.0

82/68

LEU

CTG

HIS

19

CAC

2.2

82/68

LEU

CTG

ILE

19

ATC

0.8

82/68

LEU

CTG

MET

19

ATG

1.1

82/68

LEU

CTG

PHE

19

TTC

3.2

82/68

LEU

CTG

SER

19

TCC

2.2

82/68

LEU

CTG

THR

19

ACC

1.6

82/68

LEU

CTG

TYR

19

TAC

2.7

94/80

ARG

CGA

GLN

19

CAG

0.03

94/80

ARG

CGA

HIS

19

CAC

0.01

94/80

ARG

CGA

LYS

19

AAA

*

95/81

HIS

CAT

ASN

19

AAC

2.7 (2) 2.3

95/81

HIS

CAT

ILE

19

ATC

0.33

95/81

HIS

CAT

LYS

19

AAA

0.9

95/81

HIS

CAT

MET

19

ATG

1

95/81

HIS

CAT

PHE

19

TTC

0.66

95/81

HIS

CAT

SER

19

TCC

4

95/81

HIS

CAT

TRP

19

TGG

*

98/84

HIS

CAT

ARG

19

CGT

3.2

98/84

HIS

CAT

GLN

19

CAA

2.2

98/84

HIS

CAT

GLU

19

GAA

1.55 (2) 0.15

98/84

HIS

CAT

LYS

19

AAA

4

98/84

HIS

CAT

MET

19

ATG

2.2

98/84

HIS

CAT

PHE

19

TTC

1

98/84

HIS

CAT

SER

19

TCC

4

98/84

HIS

CAT

THR

19

2.2

98/84

HIS

CAT

VAL

19

GTA

2.4 (2) 0.8

101/87

ASP

GAC

ASN

19

AAC

7

101/87

ASP

GAC

GLU

19

GAA

*

101/87

ASP

GAC

ILE

19

ATC

3.2

101/87

ASP

GAC

LEU

19

CTG

3.2

108/94

ARG

CGG

ALA

19

GCT

4

108/94

ARG

CGG

GLN

19

CAG

0.4

108/94

ARG

CGG

HIS

19

CAC

*

108/94

ARG

CGG

SER

19

TCC

3.7

110/96

LYS

AAA

GLU

19

GAA

*

110/96

LYS

AAA

HIS

19

CAC

*

110/96

LYS

AAA

ILE

19

ATC

*

113/99

PHE

TTC

ASP

19

GAC

*

113/99

PHE

TTC

ILE

19

ATC

*

113/99

PHE

TTC

LEU

19

CTG

*

113/99

PHE

TTC

LYS

19

AAA

*

116/102

LYS

AAA

ALA

19

GCT

5

116/102

LYS

AAA

ARG

19

CGT

0.03

116/102

LYS

AAA

ASN

19

AAC

0.22

116/102

LYS

AAA

GLN

19

CAG

0.33

116/102

LYS

AAA

HIS

19

CAC

3.2

116/102

LYS

AAA

MET

19

ATG

0.9

116/102

LYS

AAA

PHE

19

TTC

2.5

116/102

LYS

AAA

TYR

19

TAC

5.4 (2) 0.3

549

23 base pairs

nucleic acid

single

linear

DNA (synthetic)

1
CTAGCGATCT TTTAATAAGC TTG 23

23 base pairs

nucleic acid

single

linear

DNA (synthetic)

2
GATCCAAGCT TATTAAAAGA TCG 23

69 base pairs

nucleic acid

single

linear

DNA (synthetic)

3
GGCAACAATT TCTACAAAAC ACTTGATACT GTATGAGCAT ACAGTATAAT TGCTTCAACA 60
GAACAGATC 69

67 base pairs

nucleic acid

single

linear

DNA (synthetic)

4
TGTTCTGTTG AAGCAATTAT ACTGTATGCT CATACAGTAT CAAGTGTTTT GTAGAAATTG 60
TTGCCGC 67

23 base pairs

nucleic acid

single

linear

DNA (synthetic)

5
CCATTGCTGC CGGCATCGTG GTC 23

46 base pairs

nucleic acid

single

linear

DNA (synthetic)

6
CATGGCTCCA ATGACTCAGA CTACTTCTCT TAAGACTTCT TGGGTT 46

42 base pairs

nucleic acid

single

linear

DNA (synthetic)

7
AACCCAAGAA GTCTTAAGAG AAGTAGTCTG AGTCATTGGA GC 42

64 base pairs

nucleic acid

single

linear

DNA (synthetic)

8
AATTCCGTCG TAAACTGACC TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAGT 60
AATA 64

64 base pairs

nucleic acid

single

linear

DNA (synthetic)

9
AGCTTATTAC TGTTGAGCCT GCGCGTTCTC CAAGGTTTTC AGATAGAAGG TCAGTTTACG 60
ACGG 64

126 amino acids

amino acid

linear

peptide

10
Met Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn
1 5 10 15
Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro
20 25 30
Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile
35 40 45
Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg
50 55 60
Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys
65 70 75 80
Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His
85 90 95
Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu
100 105 110
Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
115 120 125

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

11
CATGGCTAAC TGCTCTAACA TGAT 24

22 base pairs

nucleic acid

single

linear

DNA (synthetic)

12
CGATCATGTT AGAGCAGTTA GC 22

113 amino acids

amino acid

linear

peptide

13
Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys
1 5 10 15
Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp
20 25 30
Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala
35 40 45
Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser
50 55 60
Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro
65 70 75 80
Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg
85 90 95
Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln
100 105 110
Gln

27 amino acids

amino acid

linear

peptide

14
Met Met Ile Thr Leu Arg Lys Leu Pro Leu Ala Val Ala Val Ala Ala
1 5 10 15
Gly Val Met Ser Ala Gln Ala Met Ala Asn Cys
20 25

133 amino acids

amino acid

linear

peptide

Modified-site

/note= “Met- may or may not precede the
amino acid in position 1”

Modified-site

/note= “Xaa at position 17 is Ser,
Lys, Gly, Asp, Met, Gln, or Arg”

Modified-site

/note= “Xaa at position 18 is Asn,
His, Leu, Ile, Phe, Arg, or Gln”

Modified-site

/note= “Xaa at positiion 19 is Met,
Phe, Ile, Arg, Gly, Ala, or Cys”

Modified-site

/note= “Xaa at position 20 is Ile,
Cys, Gln, Glu, Arg, Pro, or Ala”

Modified-site

/note= “Xaa at position 21 is Asp,
Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser,
or Val”

Modified-site

/note= “Xaa at position 22 is Glu,
Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val,
or Gly”

Modified-site

/note= “Xaa at position 23 is Ile,
Val, Ala, Leu, Gly, Trp, Lys, Phe, Leu, Ser, or
Arg”

Modified-site

/note= “Xaa at position 24 is Ile,
Gly, Val, Arg, Ser, Phe, or Leu”

Modified-site

/note= “Xaa at position 25 is Thr,
His, Gly, Gln, Arg, Pro, or Ala”

Modified-site

/note= “Xaa at position 26 is His,
Thr, Phe, Gly, Arg, Ala, or Trp”

Modified-site

/note= “Xaa at position 27 is Leu,
Gly, Arg, Thr, Ser, or Ala”

Modified-site

/note= “Xaa at position 28 is Lys,
Arg, Leu, Gln, Gly, Pro, Val, or Trp”

Modified-site

/note= “Xaa at position 29 is Gln,
Asn, Leu, Pro, Arg, or Val”

Modified-site

/note= “Xaa at position 30 is Pro,
His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys”

Modified-site

/note= “Xaa at position 31 is Pro,
Asp, Gly, Ala, Arg, Leu, or Gln”

Modified-site

/note= “Xaa at position 32 is Leu,
Val, Arg, Gln, Asn, Gly, Ala, or Glu”

Modified-site

/note= “Xaa at position 33 is Pro,
Leu, Gln, Ala, Thr, or Glu”

Modified-site

/note= “Xaa at position 34 is Leu,
Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe,
Ile, or Met”

Modified-site

/note= “Xaa at position 35 is Leu,
Ala, Gly, Asn, Pro, Gln, or Val”

Modified-site

/note= “Xaa at position 36 is Asp,
Leu, or Val”

Modified-site

/note= “Xaa at position 37 is Phe,
Ser, Pro, Trp, or Ile”

Modified-site

/note= “Xaa at position 38 is Asn,
or Ala”

Modified-site

/note= “Xaa at position 40 is Leu,
Trp, or Arg”

Modified-site

/note= “Xaa at position 41 is Asn,
Cys, Arg, Leu, His, Met, or Pro”

Modified-site

/note= “Xaa at position 42 is Gly,
Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr,
Ile, Met, or Ala”

Modified-site

/note= “Xaa at position 43 is Glu,
Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly,
or Ser”

Modified-site

/note= “Xaa at position 44 is Asp,
Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala,
or Pro”

Modified-site

/note= “Xaa at position 45 is Gln,
Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg,
Ser, Ala, Ile, Glu, or His”

Modified-site

/note= “Xaa at position 46 is Asp,
Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr,
Ile, Val, or Gly”

Modified-site

/note= “Xaa at position 47 is Ile,
Gly, Val, Ser, Arg, Pro, or His”

Modified-site

/note= “Xaa at position 48 is Leu,
Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met,
Val, or Asn”

Modified-site

/note= “Xaa at position 49 is Met,
Arg, Ala, Gly, Pro, Asn, His, or Asp”

Modified-site

/note= “Xaa at position 50 is Glu,
Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His,
Phe, Met, or Gln”

Modified-site

/note= “Xaa at position 51 is Asn,
Arg, Met, Pro, Ser, Thr, or His”

Modified-site

/note= “Xaa at position 52 is Asn,
His, Arg, Leu, Gly, Ser, or Thr”

Modified-site

/note= “Xaa at position 53 is
Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met”

Modified-site

/note= “Xaa at position 54 is Arg,
Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala,
or Leu”

Modified-site

/note= “Xaa at position 55 is Arg,
Thr, Val, Ser, Leu, or Gly”

Modified-site

/note= “Xaa at position 56 is Pro,
Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr,
Phe, Leu, Val, or Lys”

Modified-site

/note= “Xaa at position 57 is Asn
or Gly”

Modified-site

/note= “Xaa at position 58 is Leu,
Ser, Asp, Arg, Gln, Val, or Cys”

Modified-site

/note= “Xaa at position 59 is Glu,
Tyr, His, Leu, Pro, or Arg”

Modified-site

/note= “Xaa at position 60 is Ala,
Ser, Pro, Tyr, Asn, or Thr”

Modified-site

/note= “Xaa at position 61 is Phe,
Asn, Glu, Pro, Lys, Arg, or Ser”

Modified-site

/note= “Xaa at position 62 is Asn,
His, Val, Arg, Pro, Thr, Asp, or Ile”

Modified-site

/note= “Xaa at position 63 is Arg,
Tyr, Trp, Lys, Ser, His, Pro, or Val”

Modified-site

/note= “Xaa at position 64 is Ala,
Asn, Pro, Ser, or Lys”

Modified-site

/note= “Xaa at position 65 is Val,
Thr, Pro, His, Leu, Phe, or Ser”

Modified-site

/note= “Xaa at position 66 is Lys,
Ile, Arg, Val, Asn, Glu, or Ser”

Modified-site

/note= “Xaa at position 67 is Ser,
Ala, Phe, Val, Gly, Asn, Ile, Pro, or His”

Modified-site

/note= “Xaa at position 68 is Leu,
Val, Trp, Ser, Ile, Phe, Thr, or His”

Modified-site

/note= “Xaa at position 69 is Gln,
Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu”

Modified-site

/note= “Xaa at position 70 is Asn,
Leu, Val, Trp, Pro, or Ala”

Modified-site

/note= “Xaa at position 71 is
Ala,Met,Leu,Pro,Arg,Glu,Thr,Gln,Trp,or Asn”

Modified-site

/note= “Xaa at position 72 is Ser,
Glu, Met, Ala, His, Asn, Arg, or Asp”

Modified-site

/note= “Xaa at position 73 is Ala,
Glu, Asp, Leu, Ser, Gly, Thr, or Arg”

Modified-site

/note= “Xaa at position 74 is Ile,
Met, Thr, Pro, Arg, Gly, or Ala”

Modified-site

/note= “Xaa at position 75 is
Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln,
or Leu”

Modified-site

/note= “Xaa at position 76 is Ser,
Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp”

Modified-site

/note= “Xaa at position 77 is Ile,
Ser, Arg, Thr, or Leu”

Modified-site

/note= “Xaa at position 78 is Leu,
Ala, Ser, Glu, Phe, Gly, or Arg”

Modified-site

/note= “Xaa at position 79 is Lys, Thr,
Asn, Met, Arg, Ile, Gly, or Asp”

Modified-site

/note= “Xaa at position 80 is Asn,
Trp, Val, Gly, Thr, Leu, Glu, or Arg”

Modified-site

/note= “Xaa at position 81 is Leu,
Gln, Gly, Ala, Trp, Arg, Val, or Lys”

Modified-site

/note= “Xaa at position 82 is Leu,
Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala,
Tyr, Phe, Ile, Met, or Val”

Modified-site

/note= “Xaa at position 83 is Pro,
Ala, Thr, Trp, Arg, or Met”

Modified-site

/note= “Xaa at position 84 is Cys,
Glu, Gly, Arg, Met, or Val”

Modified-site

/note= “Xaa at position 85 is Leu,
Asn, Val, or Gln”

Modified-site

/note= “Xaa at position 86 is Pro,
Cys, Arg, Ala, or Lys”

Modified-site

/note= “Xaa at position 87 is Leu,
Ser, Trp, or Gly”

Modified-site

/note= “Xaa at position 88 is Ala,
Lys, Arg, Val, or Trp”

Modified-site

/note= “Xaa at position 89 is Thr,
Asp, Cys, Leu, Val, Glu, His, Asn, or Ser”

Modified-site

/note= “Xaa at position 90 is Ala,
Pro, Ser, Thr, Gly, Asp, Ile, or Met”

Modified-site

/note= “Xaa at position 91 is Ala,
Pro, Ser, Thr, Phe, Leu, Asp, or His”

Modified-site

/note= “Xaa at position 92 is Pro,
Phe, Arg, Ser, Lys, His, Ala, Gly, Ile, or Leu”

Modified-site

/note= “Xaa at position 93 is Thr,
Asp, Ser, Asn, Pro, Ala, Leu, or Arg”

Modified-site

/note= “Xaa at position 94 is Arg,
Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro”

Modified-site

/note= “Xaa at position 95 is His,
Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala,
Trp, Phe, Ile, or Tyr”

Modified-site

/note= “Xaa at position 96 is Pro,
Lys, Tyr, Gly, Ile, or Thr”

Modified-site

/note= “Xaa at position 97 is Ile,
Val, Lys, Ala, or Asn”

Modified-site

/note= “Xaa at position 98 is His,
Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met,
Val, Lys, Arg, Tyr, or Pro”

Modified-site

/note= “Xaa at position 99 is Ile,
Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe,
or His”

Modified-site

100

/note= “Xaa at position 100 is
Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro”

Modified-site

101

/note= “Xaa at position 101 is
Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser,
Ala, Gly, Ile, Leu, or Gln”

Modified-site

102

/note= “Xaa at position 102 is Gly,
Leu, Glu, Lys, Ser, Tyr, or Pro”

Modified-site

103

/note= “Xaa at position 103 is Asp,
or Ser”

Modified-site

104

/note= “Xaa at position 104 is
Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala,
Phe, or Gly”

Modified-site

105

/note= “Xaa at position 105 is
Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile,
Asp, or His”

Modified-site

106

/note= “Xaa at position 106 is Glu,
Ser, Ala, Lys, Thr, Ile, Gly, or Pro”

Modified-site

108

/note= “Xaa at position 108 is Arg,
Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro”

Modified-site

109

/note= “Xaa at position 109 is Arg,
Thr, Pro, Glu, Tyr, Leu, Ser, or Gly”

Modified-site

110

/note= “Xaa at position 110 is Lys,
Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, Ala,
or Trp”

Modified-site

111

/note= “Xaa at position 111 is Leu,
Ile, Arg, Asp, or Met”

Modified-site

112

/note= “Xaa at position 112 is Thr,
Val, Gln, Tyr, Glu, His, Ser, or Phe”

Modified-site

113

/note= “Xaa at position 113 is Phe,
Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val,
or Asn”

Modified-site

114

/note= “Xaa at position 114 is Tyr,
Cys, His, Ser, Trp, Arg, or Leu”

Modified-site

115

/note= “Xaa at position 115 is
Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or
Met”

Modified-site

116

/note= “Xaa at position 116 is Lys,
Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser,
Asn, His, Ala, Tyr, Phe, Gln, or Ile”

Modified-site

117

/note= “Xaa at position 117 is Thr,
Ser, Asn, Ile, Trp, Lys, or Pro”

Modified-site

118

/note= “Xaa at position 118 is Leu,
Ser, Pro, Ala, Glu, Cys, Asp, or Tyr”

Modified-site

119

/note= “Xaa at position 119 is Glu,
Ser, Lys, Pro, Leu, Thr, Tyr, or Arg”

Modified-site

120

/note= “Xaa at position 120 is Asn,
Ala, Pro, Leu, His, Val, or Gln”

Modified-site

121

/note= “Xaa at position 121 is Ala,
Ser, Ile, Asn, Pro, Lys, Asp, or Gly”

Modified-site

122

/note= “Xaa at position 122 is
Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr,
or Cys”

Modified-site

123

/note= “Xaa at position 123 is Ala,
Met, Glu, His, Ser, Pro, Tyr, or Leu”

15
Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
65 70 75 80
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
85 90 95
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa
100 105 110
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln Gln Thr Thr Leu
115 120 125
Ser Leu Ala Ile Phe
130

133 amino acids

amino acid

linear

peptide

Modified-site

/note= “Met- may or may not precede
the amino acid in position 1”

Modified-site

/note= “Xaa at position 17 is Ser,
Gly, Asp, Met, or Gln”

Modified-site

/note= “Xaa at position 18 is Asn,
His, Leu, Ile, Phe, Arg, or Gln”

Modified-site

/note= “Xaa at position 19 is Met,
Phe, Ile, Arg, or Ala”

Modified-site

/note= “Xaa at position 20 is Ile
or Pro”

Modified-site

/note; “Xaa at position 21 is Asp
or Glu”

Modified-site

/note= “Xaa at position 23 is Ile,
Val, Ala, Leu, or Gly”

Modified-site

/note= “Xaa at position 24 is Ile,
Val, Phe, or Leu”

Modified-site

/note= “Xaa at position 25 is Thr,
His, Gly, Gln, Arg, Pro, or Ala”

Modified-site

/note= “Xaa at position 26 is His,
Phe, Gly, Arg, or Ala”

Modified-site

/note= “Xaa at position 28 is Lys,
Leu, Gln, Gly, Pro, or Val”

Modified-site

/note= “Xaa at position 29 is Gln,
Asn, Leu, Arg, or Val”

Modified-site

/note= “Xaa at position 30 is Pro,
His, Thr, Gly, or Gln”

Modified-site

/note= “Xaa at position 31 is Pro,
Asp, Gly, Ala, Arg, Leu, or Gln”

Modified-site

/note= “Xaa at position 32 Leu,
Arg, Gln, Asn, Gly, Ala, or Glu”

Modified-site

/note= “Xaa at position 33 is Pro,
Leu, Gln, Ala, or Glu”

Modified-site

/note= “Xaa at position 34 is Leu,
Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe,
Thr, or Met”

Modified-site

/note= “Xaa at position 35 is Leu,
Ala, Asn, Pro, Gln, or Val”

Modified-site

/note= “Xaa at position 36 is Asp
or Leu”

Modified-site

/note= “Xaa at position 37 is Phe,
Ser, Pro, Trp, or Ile”

Modified-site

/note= “Xaa at position 38 is Asn
or Ala”

Modified-site

/note= “Xaa at position 41 is Asn,
Cys, Arg, His, Met, or Pro”

Modified-site

/note= “Xaa at position 42 is Gly,
Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, Val,
or Arg”

Modified-site

/note=“Xaa at position 44 is Asp or
Glu”

Modified-site

/note= “Xaa at position 45 is Gln,
Val, Met, Leu, Thr, Lys, Ala, Asn, Glu, Ser, or Trp”

Modified-site

/note= “Xaa at position 46 is Asp,
Phe, Ser, Thr, Cys, Ala, Asn, Gln, Glu, His, Ile, Lys,
Tyr, Val, or Gly”

Modified-site

/note= “Xaa at position 47 is Ile, Val,
or His”

Modified-site

/note= “Xaa at position 49 is Met,
Asn, or Asp”

Modified-site

/note= “Xaa at position 50 is Glu,
Thr, Ala, Asn, Ser, or Asp”

Modified-site

/note= “Xaa at position 51 is Asn,
Arg, Met, Pro, Ser, Thr, or His”

Modified-site

/note= “Xaa at position 52 is Asn
or Gly”

Modified-site

/note= “Xaa at position 53 is Leu,
Met, or Phe”

Modified-site

/note= “Xaa at position 54 is Arg
Ala, or Ser”

Modified-site

/note= “Xaa at position 55 is Arg,
Thr, Val, Leu, or Gly”

Modified-site

/note= “Xaa at position 56 is Pro,
Gly, Cys, Ser, Gln, Ala, Arg, Asn, Glu, His, Leu, Thr,
Val, or Lys”

Modified-site

/note= “Xaa at position 59 is Glu,
Tyr, His, Leu, or Arg”

Modified-site

/note= “Xaa at position 60 is Ala,
Ser, Asn, or Thr”

Modified-site

/note= “Xaa at position 61 is Phe
or Ser”

Modified-site

/note= “Xaa at position 62 is Asn,
Val, Pro, Thr, or Ile”

Modified-site

/note= “Xaa at position 63 is Arg,
Tyr, Lys, Ser, His, or Val”

Modified-site

/note= “Xaa at position 64 is Ala
or Asn”

Modified-site

/note= “Xaa at position 65 is Val,
Thr, Leu, or Ser”

Modified-site

/note= “Xaa at position 66 is Lys,
Ile, Arg, Val, Asn, Glu, or Ser”

Modified-site

/note= “Xaa at position 67 is Ser,
Phe, Val, Gly, Asn, Ile, or His”

Modified-site

/note= “Xaa at position 68 is Leu,
Val, Ile, Phe, or His”

Modified-site

/note= “Xaa at position 69 is Gln,
Ala, Pro, Thr, Glu, Arg, or Gly”

Modified-site

/note= “Xaa at position 70 is Asn
or Pro”

Modified-site

/note= “Xaa at position 71 is Ala,
Met, Pro, Arg, Glu, Thr, or Gln”

Modified-site

/note= “Xaa at position 72 is Ser,
Glu, Met, Ala, His, Asn, Arg, or Asp”

Modified-site

/note= “Xaa at position 73 is Ala,
Glu, Asp, Leu, Ser, Gly, Thr, Arg, or Pro”

Modified-site

/note= “Xaa at position 74 is Ile
or Met”

Modified-site

/note= “Xaa at position 75 is Glu,
Gly, Asp, Ser, or Gln”

Modified-site

/note= “Xaa at position 76 is Ser,
Val, Ala, Asn, Glu, Pro, Gly, or Asp”

Modified-site

/note= “Xaa at position 77 is Ile,
Ser, or Leu”

Modified-site

/note= “Xaa at position 79 is Lys,
Thr, Gly, Asn, Met, Arg, Ile, Gly, or Asp”

Modified-site

/note= “Xaa at position 80 is Asn,
Val, Gly, Thr, Leu, Glu, or Arg”

Modified-site

/note= “Xaa at position 81 is Leu
or Val”

Modified-site

/note= “Xaa at position 82 is Leu,
Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Met, Phe,
Ser, Thr, Tyr, or Val”

Modified-site

/note= “Xaa at position 83 is Pro,
Ala, Thr, Trp, or Met”

Modified-site

/note= “Xaa at position 85 is Leu
or Val”

Modified-site

/note= “Xaa at position 87 is Leu
or Ser”

Modified-site

/note= “Xaa at position 88 is Ala,
Arg, or Trp”

Modified-site

/note= “Xaa at position 89 is Thr,
Asp, Glu, His, Asn, or Ser”

Modified-site

/note= “Xaa at position 90 is Ala,
Asp, or Met”

Modified-site

/note= “Xaa at position 91 is Ala,
Pro, Ser, Thr, Phe, Leu, or Asp”

Modified-site

/note= “Xaa at position 92 is Pro
or Ser”

Modified-site

/note= “Xaa at position 93 is Thr,
Asp, Ser, Pro, Ala, Leu, or Arg”

Modified-site

/note= “Xaa at position 95 is His,
Pro, Arg, Val, Leu, Gly, Asn, Ile, Phe, Ser,
or Thr”

Modified-site

/note= “Xaa at position 96 is Pro
or Tyr”

Modified-site

/note= “Xaa at position 97 is Ile,
Val, or Ala”

Modified-site

/note= “Xaa at position 98 is His,
Ile, Asn, Leu, Asp, Ala, Thr, Leu, Arg, Gln, Glu,
Lys, Met, Ser, Tyr, Val, or Pro”

Modified-site

/note= “Xaa at position 99 is Ile,
Leu, Val, or Phe”

Modified-site

100

/note= “Xaa at position 100 is Lys,
Leu, His, Arg, Ile, Gln, Pro, or Ser”

Modified-site

101

/note= “Xaa at position 101 is Asp,
Pro, Met, Lys, His, Thr, Val, Asn, Ile, Leu, or Tyr”

Modified-site

102

/note= “Xaa at position 102 is Gly,
Glu, Lys, or Ser”

Modified-site

104

/note= “Xaa at position 104 is Trp,
Val, Tyr, Met, or Leu”

Modified-site

105

/note= “Xaa at position 105 is
Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys,
Ile, Asp or His”

Modified-site

106

/note= “Xaa at position 106 is Glu,
Ser, Ala, or Gly”

Modified-site

108

/note= “Xaa at position 108 is Arg,
Ala, Gln, Ser, or Lys”

Modified-site

109

/note= “Xaa at position 109 is Arg,
Thr, Glu, Leu, Ser, or Gly”

Modified-site

112

/note= “Xaa at position 112 is Thr,
Val, Gln, Glu, His, or Ser”

Modified-site

114

/note= “Xaa at position 114 is Tyr
or Trp”

Modified-site

115

/note= “Xaa at position 115 is Leu
or Ala”

Modified-site

116

/note= “Xaa at position 116 is Lys,
Thr, Met, Val, Trp, Ser, Leu, Ala, Asn, Gln, His, Met,
Phe, Tyr, or Ile”

Modified-site

117

/note= “Xaa at position 117 is Thr,
Ser, or Asn”

Modified-site

119

/note= “Xaa at position 119 is
Glu, Ser, Pro, Leu, Thr, or Tyr”

Modified-site

120

/note= “Xaa at position 120 is Asn,
Pro, Leu, His, Val, or Gln”

Modified-site

121

/note= “Xaa at position 121 is Ala,
Ser, Ile, Asn, Pro, Lys, Asp, or Gly”

Modified-site

122

/note= “Xaa at position 122 is
Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr,
or Cys”

Modified-site

123

/note= “Xaa at position 123 is Ala,
Met, Glu, His, Ser, Pro, Tyr, or Leu”

16
Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa
20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa
65 70 75 80
Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Xaa
85 90 95
Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Phe Xaa Xaa Lys Leu Xaa
100 105 110
Phe Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Gln Gln Thr Thr Leu
115 120 125
Ser Leu Ala Ile Phe
130

133 amino acids

amino acid

linear

peptide

Modified-site

/note= “Met- may or may not precede
the amino acid in position 1”

Modified-site

/note= “Xaa at position 17 is Ser,
Gly, Asp, Met, or Gln”

Modified-site

/note= “Xaa at position 18 is Asn,
His, or Ile”

Modified-site

/note= “Xaa at position 19 is Met
or Ile”

Modified-site

/note= “Xaa at position 21 is Asp
or Glu”

Modified-site

/note= “Xaa at position 23 is Ile,
Ala, Leu, or Gly”

Modified-site

/note= “Xaa at position 24 is Ile,
Val, or Leu”

Modified-site

/note= “Xaa at position 25 is Thr,
His, Gln, or Ala”

Modified-site

/note= “Xaa at position 26 is His
or Ala”

Modified-site

/note= “Xaa at position 29 is Gln,
Asn, or Val”

Modified-site

/note= “Xaa at position 30 is Pro,
Gly, or Gln”

Modified-site

/note= “Xaa at position 31 is Pro,
Asp, Gly, or Gln”

Modified-site

/note= “Xaa at position 32 is Leu,
Arg, Gln, Asn, Gly, Ala, or Glu”

Modified-site

/note= “Xaa at position 33 is Pro
or Glu”

Modified-site

/note= “Xaa at position 34 is Leu,
Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe
Thr, or Met”

Modified-site

/note= “Xaa at position 35 is Leu,
Ala, Asn, Pro, Gln, or Val”

Modified-site

/note= “Xaa at position 37 is Phe,
Ser, Pro, or Trp”

Modified-site

/note= “Xaa at position 38 is Asn
or Ala”

Modified-site

/note= “Xaa at position 42 is Gly,
Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, or Arg”

Modified-site

/note= “Xaa at position 45 is Gln,
Val, Met, Leu, Thr, Ala, Asn, Glu, Ser, or Lys”

Modified-site

/note= “Xaa at position 46 is Asp,
Phe, Ser, Thr, Ala, Asn, Gln, Glu, His, Ile, Lys,
Tyr, Val, or Cys”

Modified-site

/note= “Xaa at position 50 is Glu,
Ala, Asn, Ser, or Asp”

Modified-site

/note= “Xaa at position 51 is Asn,
Arg, Met, Pro, Ser, Thr, or His”

Modified-site

/note= “Xaa at position 54 is Arg
or Ala”

Modified-site

/note= “Xaa at position 55 is Arg,
Thr, Val, Leu, or Gly”

Modified-site

/note= “Xaa at position 56 is Pro,
Gly, Ser, Gln, Ala, Arg, Asn, Glu, Leu, Thr, Val,
or Lys”

Modified-site

/note= “Xaa at position 60 is Ala
or Ser”

Modified-site

/note= “Xaa at position 62 is Asn,
Pro, Thr, or Ile”

Modified-site

/note= “Xaa at position 63 is Arg
or Lys”

Modified-site

/note= “Xaa at position 64 is Ala
or Asn”

Modified-site

/note= “Xaa at position 65 is Val
or Thr”

Modified-site

/note= “Xaa at position 66 is Lys
or Arg”

Modified-site

/note= “Xaa at position 67 is Ser
Phe or His”

Modified-site

/note= “Xaa at position 68 is Leu,
Ile, Phe, or His”

Modified-site

/note= “Xaa at position 69 is Gln,
Ala, Pro, Thr, Glu, Arg, or Gly”

Modified-site

/note= “Xaa at position 71 is Ala,
Pro, or Arg”

Modified-site

/note= “Xaa at position 72 is Ser,
Glu, Arg, or Asp”

Modified-site

/note= “Xaa at position 73 is Ala
or Leu”

Modified-site

/note= “Xaa at position 76 is Ser,
Val, Ala, Asn, Glu, Pro, or Gly”

Modified-site

/note= “Xaa at position 77 is Ile
or Leu”

Modified-site

/note= “Xaa at position 79 is
Lys, Thr, Gly, Asn, Met, Arg, Ile, Gly, or Asp”

Modified-site

/note= “Xaa at position 80 is Asn,
Gly, Glu, or Arg”

Modified-site

/note= “Xaa at position 82 is Leu,
Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Ile, Met,
Phe, Ser, Thr, Tyr, or Val”

Modified-site

/note= “Xaa at position 83 is Pro
or Thr”

Modified-site

/note= “Xaa at position 85 is Leu
or Val”

Modified-site

/note= “Xaa at position 87 is Leu
or Ser”

Modified-site

/note= “Xaa at position 88 is Ala
or Trp”

Modified-site

/note= “Xaa at position 91 is Ala
or Pro”

Modified-site

/note= “Xaa at position 93 is Thr,
Asp, Ser, Pro, Ala, Leu, or Arg”

Modified-site

/note= “Xaa at position 95 is His,
Pro, Arg, Val, Leu, Gly, Asn, Phe, Ser, or Thr”

Modified-site

/note= “Xaa at position 96 is Pro
or Tyr”

Modified-site

/note= “Xaa at position 97 is Ile
or Val”

Modified-site

/note= “Xaa at position 98 is His,
Ile, Asn, Leu, Ala, Thr, Leu, Arg, Gln, Leu, Lys,
Met, Ser, Tyr, Val, or Pro”

Modified-site

/note= “Xaa at position 99 is Ile,
Leu, or Val”

Modified-site

100

/note= “Xaa at position 100 is Lys,
Arg, Ile, Gln, Pro, or Ser”

Modified-site

101

/note= “Xaa at position 101 is Asp,
Pro, Met, Lys, His, Thr, Pro, Asn, Ile, Leu, or Tyr”

Modified-site

104

/note= “Xaa at position 104 is Trp
or Leu”

Modified-site

105

/note= “Xaa at position 105 is
Asn, Pro, Ala, Ser, Trp, Gln, Tyr, Leu, Lys, Ile,
Asp, or His”

Modified-site

106

/note= “Xaa at position 106 is Glu
or Gly”

Modified-site

109

/note= “Xaa at position 109 is Arg,
Thr, Glu, Leu, or Ser”

Modified-site

112

/note= “Xaa at position 112 is Thr,
Val, or Gln”

Modified-site

114

/note= “Xaa at position 114 is Tyr
or Trp”

Modified-site

115

/note= “Xaa at position 115 is Leu
or Ala”

Modified-site

116

/note= “Xaa at position 116 is Lys,
Thr, Val, Trp, Ser, Ala, His, Met, Phe, Tyr, or Ile”

Modified-site

117

/note= “Xaa at position 117 is Thr
or Ser”

Modified-site

120

/note= “Xaa at position 120 is Asn,
Pro, Leu, His, Val, or Gln”

Modified-site

121

/note= “Xaa at position 121 is Ala,
Ser, Ile, Asn, Pro, Asp, or Gly”

Modified-site

122

/note= “Xaa at position 122 is
Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr,
or Cys”

Modified-site

123

/note= “Xaa at position 123 is Ala,
Met, Glu, His, Ser, Pro, Tyr, or Leu”

17
Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys
1 5 10 15
Xaa Xaa Xaa Ile Xaa Glu Xaa Xaa Xaa Xaa Leu Lys Xaa Xaa Xaa Xaa
20 25 30
Xaa Xaa Xaa Asp Xaa Xaa Asn Leu Asn Xaa Glu Xaa Xaa Xaa Ile Leu
35 40 45
Met Xaa Xaa Asn Leu Xaa Xaa Xaa Asn Leu Glu Xaa Phe Xaa Xaa Xaa
50 55 60
Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Ile Glu Xaa Xaa Leu Xaa Xaa
65 70 75 80
Leu Xaa Xaa Cys Xaa Pro Xaa Xaa Thr Ala Xaa Pro Xaa Arg Xaa Xaa
85 90 95
Xaa Xaa Xaa Xaa Xaa Gly Asp Xaa Xaa Xaa Phe Xaa Xaa Lys Leu Xaa
100 105 110
Phe Xaa Xaa Xaa Xaa Leu Glu Xaa Xaa Xaa Xaa Gln Gln Thr Thr Leu
115 120 125
Ser Leu Ala Ile Phe
130

133 amino acids

amino acid

linear

peptide

Modified-site

/note= “Met- may or may not precede
the amino acid in position 1”

Modified-site

/note= “Xaa at position 17 is Ser,
Gly, Asp, or Gln”

Modified-site

/note= “Xaa at position 18 is Asn,
His, or Ile”

Modified-site

/note= “Xaa at position 23 is Ile,
Ala, Leu, or Gly”

Modified-site

/note= “Xaa at position 25 is Thr,
His, or Gln”

Modified-site

/note= “Xaa at position 26 is His
or Ala”

Modified-site

/note=“Xaa at position 29 is Gln
or Asn”

Modified-site

/note= “Xaa at position 30 is Pro
or Gly”

Modified-site

/note= “Xaa at position 32 is Leu,
Arg, Asn, or Ala”

Modified-site

/note= “Xaa at position 34 is Leu,
Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr, or Met”

Modified-site

/note= “Xaa at position 35 is Leu,
Ala, Asn, or Pro”

Modified-site

/note= “Xaa at position 38 is Asn
or Ala”

Modified-site

/note= “Xaa at position 42 is Gly,
Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr, or Arg”

Modified-site

/note= “Xaa at position 45 is Gln,
Val, Met, Leu, Ala, Asn, Glu, or Lys”

Modified-site

/note= “Xaa at position 46 is Asp,
Phe, Ser, Ala, Gln, Glu, His, Val, or Thr”

Modified-site

/note= “Xaa at position 50 is Glu,
Asn, Ser, or Asp”

Modified-site

/note= “Xaa at position 51 is Asn,
Arg, Pro, Thr, or His”

Modified-site

/note= “Xaa at position 55 is Arg,
Leu, or Gly”

Modified-site

/note= “Xaa at position 56 is Pro,
Gly, Ser, Ala, Asn, Val, Leu, or Gln”

Modified-site

/note= “Xaa at position 62 is Asn,
Pro, or Thr”

Modified-site

/note= “Xaa at position 64 is Ala
or Asn”

Modified-site

/note= “Xaa at position 65 is Val
or Thr”

Modified-site

/note= “Xaa at position 67 is Ser
or Phe”

Modified-site

/note= “Xaa at position 68 is Leu
or Phe”

Modified-site

/note= “Xaa at position 69 is Gln,
Ala, Glu, or Arg”

Modified-site

/note= “Xaa at position 76 is Ser,
Val, Asn, Pro, or Gly”

Modified-site

/note= “Xaa at position 77 is Ile
or Leu”

Modified-site

/note= “Xaa at position 79 is Lys,
Gly, Asn, Met, Arg, Ile, or Gly”

Modified-site

/note= “Xaa at position 80 is Asn,
Gly, Glu, or Arg”

Modified-site

/note= “Xaa at position 82 is Leu,
Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser,
Thr, Tyr, or Val”

Modified-site

/note= “Xaa at position 87 is Leu
or Ser”

Modified-site

/note= “Xaa at position 88 is Ala
or Trp”

Modified-site

/note= “Xaa at position 91 is Ala
or Pro”

Modified-site

/note= “Xaa at position 93 is Thr,
Asp, or Ala”

Modified-site

/note= “Xaa at position 95 is His,
Pro, Arg, Val, Gly, Asn, Ser, or Thr”

Modified-site

/note= “Xaa at position 98 is His,
Ile, Asn, Ala, Thr, Arg, Gln, Glu, Lys, Met, Ser,
Tyr, Val, or Leu”

Modified-site

/note= “Xaa at position 99 is Ile
or Leu”

Modified-site

100

/note= “Xaa at position 100 is Lys
or Arg”

Modified-site

101

/note= “Xaa at position 101 is Asp,
Pro, Met, Lys, Thr, His, Pro, Asn, Ile, Leu, or Tyr”

Modified-site

105

/note= “Xaa at position 105 is Asn,
Pro, Ser, Ile, or Asp”

Modified-site

108

/note= “Xaa at position 108 is Arg,
Ala, or Ser”

Modified-site

109

/note= “Xaa at position 109 is Arg,
Thr, Glu, Leu, or Ser”

Modified-site

112

/note= “Xaa at position 112 is Thr
or Gln”

Modified-site

116

/note= “Xaa at position 116 is Lys,
Val, Trp, Ala, His, Phe, Tyr, or Ile”

Modified-site

117

/note= “Xaa at position 117 is Thr
or Ser”

Modified-site

120

/note= “Xaa at position 120 is Asn,
Pro, Leu, His, Val, or Gln”

Modified-site

121

/note= “Xaa at position 121 is Ala,
Ser, Ile, Pro, or Asp”

Modified-site

122

/note= “Xaa at position 122 is Gln,
Met, Trp, Phe, Pro, His, Ile, or Tyr”

Modified-site

123

/note= “Xaa at position 123 is Ala,
Met, Glu, Ser, or Leu”

18
Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys
1 5 10 15
Xaa Xaa Met Ile Asp Glu Xaa Ile Xaa Xaa Leu Lys Xaa Xaa Pro Xaa
20 25 30
Pro Xaa Xaa Asp Phe Xaa Asn Leu Asn Xaa Glu Asp Xaa Xaa Ile Leu
35 40 45
Met Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Glu Ala Phe Xaa Arg Xaa
50 55 60
Xaa Lys Xaa Xaa Xaa Asn Ala Ser Ala Ile Glu Xaa Xaa Leu Xaa Xaa
65 70 75 80
Leu Xaa Pro Cys Leu Pro Xaa Xaa Thr Ala Xaa Pro Xaa Arg Xaa Pro
85 90 95
Ile Xaa Xaa Xaa Xaa Gly Asp Trp Xaa Glu Phe Xaa Xaa Lys Leu Xaa
100 105 110
Phe Tyr Leu Xaa Xaa Leu Glu Xaa Xaa Xaa Xaa Gln Gln Thr Thr Leu
115 120 125
Ser Leu Ala Ile Phe
130

111 amino acids

amino acid

linear

peptide

Modified-site

/note= “Met- or Met-Ala- may or may
not precede the amino acid in position 1”

Modified-site

/note= “Xaa at position 3 is Ser,
Lys, Gly, Asp, Met, Gln, or Arg”

Modified-site

/note= “Xaa at position 4 is Asn,
His, Leu, Ile, Phe, Arg, or Gln”

Modified-site

/note= “Xaa at position 5 is Met,
Phe, Ile, Arg, Gly, Ala, or Cys”

Modified-site

/note= “Xaa at position 6 is Ile,
Cys, Gln, Glu, Arg, Pro, or Ala”

Modified-site

/note= “Xaa at position 7 is Asp,
Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser,
or Val”

Modified-site

/note= “Xaa at position 8 is Glu,
Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val,
or Gly”

Modified-site

/note= “Xaa at position 9 is
Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Leu, Ser
or Arg”

Modified-site

/note= “Xaa at position 10 is Ile,
Gly, Val, Arg, Ser, Phe, or Leu”

Modified-site

/note= “Xaa at position 11 is Thr,
His, Gly, Gln, Arg, Pro, or Ala”

Modified-site

/note= “Xaa at position 12 is His,
Thr, Phe, Gly, Arg, Ala, or Trp”

Modified-site

/note= “Xaa at position 13 is Leu,
Gly, Arg, Thr, Ser, or Ala”

Modified-site

/note= “Xaa at position 14 is Lys,
Arg, Leu, Gln, Gly, Pro, Val, or Trp”

Modified-site

/note= “Xaa at position 15 is Gln,
Asn, Leu, Pro, Arg, or Val”

Modified-site

/note= “Xaa at position 16 is Pro,
His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys”

Modified-site

/note= “Xaa at position 17 is Pro,
Asp, Gly, Ala, Arg, Leu, or Gln”

Modified-site

/note= “Xaa at position 18 is Leu,
Val, Arg, Gln, Asn, Gly, Ala, or Glu”

Modified-site

/note= “Xaa at position 19 is Pro,
Leu, Gln, Ala, Thr, or Glu”

Modified-site

/note= “Xaa at position 20 is Leu,
Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe,
Ile, or Met”

Modified-site

/note= “Xaa at position 21 is Leu,
Ala, Gly, Asn, Pro, Gln, or Val”

Modified-site

/note= “Xaa at position 22 is Asp,
Leu, or Val”

Modified-site

/note= “Xaa at position 23 is Phe,
Ser, Pro, Trp, or Ile”

Modified-site

/note= “Xaa at position 24 is Asn
or Ala”

Modified-site

/note= “Xaa at position 26 is Leu,
Trp, or Arg”

Modified-site

/note= “Xaa at position 27 is Asn,
Cys, Arg, Leu, His, Met, or Pro”

Modified-site

/note= “Xaa at position 28 is Gly,
Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu,
Phe, Tyr, Ile, or Met”

Modified-site

/note= “Xaa at position 29 is Glu,
Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr,
Gly, or Ser”

Modified-site

/note= “Xaa at position 30 is Asp,
Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln,
Ala, or Pro”

Modified-site

/note= “Xaa at position 31 is Gln,
Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg,
Ser, Ala, Ile, Glu, His, or Trp”

Modified-site

/note= “Xaa at position 32 is Asp,
Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala,
Tyr, Ile, Val, or Gly”

Modified-site

/note= “Xaa at position 33 is Ile,
Gly, Val, Ser, Arg, Pro, or His”

Modified-site

/note= “Xaa at position 34 is Leu,
Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala,
Met, Val, or Asn”

Modified-site

/note= “Xaa at position 35 is Met,
Arg, Ala, Gly, Pro, Asn, His, or Asp”

Modified-site

/note= “Xaa at position 36 is Glu,
Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val,
His, Phe, Met, or Gln”

Modified-site

/note= “Xaa at position 37 is Asn,
Arg, Met, Pro, Ser, Thr, or His”

Modified-site

/note= “Xaa at position 38 is Asn,
His, Arg, Leu, Gly, Ser, or Thr”

Modified-site

/note= “Xaa at position 39 is
Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met”

Modified-site

/note= “Xaa at position 40 is Arg,
Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His,
Ala, or Leu”

Modified-site

/note= “Xaa at position 41 is Arg,
Thr, Val, Ser, Leu, or Gly”

Modified-site

/note= “Xaa at position 42 is Pro,
Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr,
Phe, Leu, Val, or Lys”

Modified-site

/note= “Xaa at position 43 is Asn
or Gly”

Modified-site

/note= “Xaa at position 44 is Leu,
Ser, Asp, Arg, Gln, Val, or Cys”

Modified-site

/note= “Xaa at position 45 is Glu,
Tyr, His, Leu, Pro, or Arg”

Modified-site

/note= “Xaa at position 46 is Ala,
Ser, Pro, Tyr, Asn, or Thr”

Modified-site

/note= “Xaa at position 47 is Phe,
Asn, Glu, Pro, Lys, Arg, or Ser”

Modified-site

/note= “Xaa at position 48 is Asn,
His, Val, Arg, Pro, Thr, Asp, or Ile”

Modified-site

/note= “Xaa at position 49 is Arg,
Tyr, Trp, Lys, Ser, His, Pro, or Val”

Modified-site

/note= “Xaa at position 50 is Ala,
Asn, Pro, Ser, or Lys”

Modified-site

/note= “Xaa at position 51 is Val,
Thr, Pro, His, Leu, Phe, or Ser”

Modified-site

/note= “Xaa at position 52 is Lys,
Ile, Arg, Val, Asn, Glu, or Ser”

Modified-site

/note= “Xaa at position 53 is Ser,
Ala, Phe, Val, Gly, Asn, Ile, Pro, or His”

Modified-site

/note= “Xaa at position 54 is Leu,
Val, Trp, Ser, Ile, Phe, Thr, or His”

Modified-site

/note= “Xaa at position 55 is Gln,
Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu”

Modified-site

/note= “Xaa at position 56 is Asn,
Leu, Val, Trp, Pro, or Ala”

Modified-site

/note= “Xaa at position 57 is Ala,
Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn”

Modified-site

/note= “Xaa at position 58 is Ser,
Glu, Met, Ala, His, Asn, Arg, or Asp”

Modified-site

/note= “Xaa at position 59 is Ala,
Glu, Asp, Leu, Ser, Gly, Thr, or Arg”

Modified-site

/note= “Xaa at position 60 is Ile,
Met, Thr, Pro, Arg, Gly, Ala”

Modified-site

/note= “Xaa at position 61 is
Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln,
or Leu”

Modified-site

/note= “Xaa at position 62 is Ser,
Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp”

Modified-site

/note= “Xaa at position 63 is Ile,
Ser, Arg, Thr, or Leu”

Modified-site

/note= “Xaa at position 64 is Leu,
Ala, Ser, Glu, Phe, Gly, or Arg”

Modified-site

/note= “Xaa at position 65 is Lys,
Thr, Gly, Asn, Met, Arg, Ile, or Asp”

Modified-site

/note= “Xaa at position 66 is Asn,
Trp, Val, Gly, Thr, Leu, Glu, or Arg”

Modified-site

/note= “Xaa at position 67 is Leu,
Gln, Gly, Ala, Trp, Arg, Val, or Lys”

Modified-site

/note= “Xaa at position 68 is Leu,
Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala,
Tyr, Phe, Ile, Met, or Val”

Modified-site

/note= “Xaa at position 69 is Pro,
Ala, Thr, Trp, Arg, or Met”

Modified-site

/note= “Xaa at position 70 is Cys,
Glu, Gly, Arg, Met, or Val”

Modified-site

/note= “Xaa at position 71 is Leu,
Asn, Val, or Gln”

Modified-site

/note= “Xaa at position 72 is Pro,
Cys, Arg, Ala, or Lys”

Modified-site

/note= “Xaa at position 73 is Leu,
Ser, Trp, or Gly”

Modified-site

/note= “Xaa at position 74 is Ala,
Lys, Arg, Val, or Trp”

Modified-site

/note= “Xaa at position 75 is Thr,
Asp, Cys, Leu, Val, Glu, His, Asn, or Ser”

Modified-site

/note= “Xaa at position 76 is Ala,
Pro, Ser, Thr, Gly, Asp, Ile, or Met”

Modified-site

/note= “Xaa at position 77 is Ala,
Pro, Ser, Thr, Phe, Leu, Asp, or His”

Modified-site

/note= “Xaa at position 78 is Pro,
Phe, Arg, Ser, Lys, His, Ala, Gly, Ile, or Leu”

Modified-site

/note= “Xaa at position 79 is Thr,
Asp, Ser, Asn, Pro, Ala, Leu, or Arg”

Modified-site

/note= “Xaa at position 80 is Arg,
Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro”

Modified-site

/note= “Xaa at position 81 is His,
Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser,
Ala, Trp, Phe, Ile, or Tyr”

Modified-site

/note= “Xaa at position 82 is Pro,
Lys, Tyr, Gly, Ile, or Thr”

Modified-site

/note= “Xaa at position 83 is Ile,
Val, Lys, Ala, or Asn”

Modified-site

/note= “Xaa at position 84 is His,
Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser,
Phe, Met, Val, Lys, Arg, Tyr, or Pro”

Modified-site

/note= “Xaa at position 85 is
Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser,
Phe, or His”

Modified-site

/note= “Xaa at position 86 is
Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro”

Modified-site

/note= “Xaa at position 87 is
Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn,
Ser, Ala, Gly, Ile, Leu, or Gln”

Modified-site

/note= “Xaa at position 88 Gly,
Leu, Glu, Lys, Ser, Tyr, or Pro”

Modified-site

/note= “Xaa at position 89 is Asp
or Ser”

Modified-site

/note= “Xaa at position 90 is
Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys,
Ala, Phe, or Gly”

Modified-site

/note= “Xaa at position 91 is
Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys,
Ile, Asp, or His”

Modified-site

/note= “Xaa at position 92 is Glu,
Ser, Ala, Lys, Thr, Ile, Gly, or Pro”

Modified-site

/note= “Xaa at position 94 is Arg,
Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro”

Modified-site

/note= “Xaa at position 95 is Arg,
Thr, Pro, Glu, Tyr, Leu, Ser, or Gly”

Modified-site

/note= “Xaa at position 96 is Lys,
Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala,
or Trp”

Modified-site

/note= “Xaa at position 97 is Leu,
Ile, Arg, Asp, or Met”

Modified-site

/note= “Xaa at position 98 is Thr,
Val, Gln, Tyr, Glu, His, Ser, or Phe”

Modified-site

/note= “Xaa at position 99 is Phe,
Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile,
Val, or Asn”

Modified-site

100

/note= “Xaa at position 100 is Tyr,
Cys, His, Ser, Trp, Arg, or Leu”

Modified-site

101

/note= “Xaa at position 101 is Leu,
Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met”

Modified-site

102

/note= “Xaa at position 102 is
Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp,
Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile”

Modified-site

103

/note= “Xaa at position 103 is Thr,
Ser, Asn, Ile, Trp, Lys, or Pro”

Modified-site

104

/note= “Xaa at position 104 is Leu,
Ser, Pro, Ala, Glu, Cys, Asp, or Tyr”

Modified-site

105

/note= “Xaa at position 105 is Glu,
Ser, Lys, Pro, Leu, Thr, Tyr, or Arg”

Modified-site

106

/note= “Xaa at position 106 is Asn,
Ala, Pro, Leu, His, Val or Gln”

Modified-site

107

/note= “Xaa at position 107 is Ala,
Ser, Ile, Asn, Pro, Lys, Asp, or Gly”

Modified-site

108

/note= “Xaa at position 108 is
Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr,
or Cys”

Modified-site

109

/note= “Xaa at position 109 is Ala,
Met, Glu, His, Ser, Pro, Tyr, or Leu”

19
Asn Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
65 70 75 80
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa
85 90 95
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln Gln
100 105 110

111 amino acids

amino acid

linear

peptide

Modified-site

/note= “Met- or Met-Ala- may or may
not precede the amino acid in position 1”

Modified-site

/note= “Xaa at position 3 is Ser,
Gly, Asp, Met, or Gln”

Modified-site

/note= “Xaa at position 4 is Asn,
His, Leu, Ile, Phe, Arg, or Gln”

Modified-site

/note= “Xaa at position 5 is Met,
Phe, Ile, Arg, or Ala”

Modified-site

/note= “Xaa at position 6 is Ile or
Pro”

Modified-site

/note= “Xaa at position 7 is Asp or
Glu”

Modified-site

/note= “Xaa at position 9 is Ile,
Val, Ala, Leu, or Gly”

Modified-site

/note= “Xaa at position 10 is Ile,
Val, Phe, or Leu”

Modified-site

/note= “Xaa at position 11 is Thr,
His, Gly, Gln, Arg, Pro, or Ala”

Modified-site

/note= “Xaa at position 12 is His,
Phe, Gly, Arg, or Ala”

Modified-site

/note= “Xaa at position 14 is Lys,
Leu, Gln, Gly, Pro, or Val”

Modified-site

/note= “Xaa at position 15 is Gln,
Asn, Leu, Arg, or Val”

Modified-site

/note= “Xaa at position 16 is Pro,
His, Thr, Gly, or Gln”

Modified-site

/note= “Xaa at position 17 is Pro,
Asp, Gly, Ala, Arg, Leu, or Gln”

Modified-site

/note= “Xaa at position 18 is Leu,
Arg, Gln, Asn, Gly, Ala or Glu”

Modified-site

/note= “Xaa at poisiton 19 is Pro,
Leu, Gln, Ala, or Glu”

Modified-site

/note= “Xaa at positon 20 is Leu,
Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe,
Thr, or Met”

Modified-site

/note= “Xaa at position 21 is Leu,
Ala, Asn, Pro, Gln, or Val”

Modified-site

/note= “Xaa at position 22 is Asp
or Leu”

Modified-site

/note= “Xaa at position 23 is Phe,
Ser, Pro, Trp, or Ile”

Modified-site

/note= “Xaa at position 24 is Asn
or Ala”

Modified-site

/note= “Xaa at position 27 is Asn,
Cys, Arg, His, Met, or Pro”

Modified-site

/note= “Xaa at position 28 is Gly,
Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, or Arg”

Modified-site

/note= “Xaa at position 30 is Asp
or Glu”

Modified-site

/note= “Xaa at position 31 is Gln,
Val, Met, Leu, Thr, Lys, Ala, Asn, Glu, Ser, or Trp”

Modified-site

/note= “Xaa at position 32 is Asp,
Phe, Ser, Thr, Cys, Ala, Asn, Gln, Glu, His, Ile,
Lys, Tyr, Val, or Gly”

Modified-site

/note= “Xaa at position 33 is Ile,
Val, or His”

Modified-site

/note= “Xaa at position 35 is Met,
Asn, or Asp”

Modified-site

/note= “Xaa at position 36 is Glu,
Thr, Ala, Asn, Ser, or Asp”

Modified-site

/note= “Xaa at position 37 is Asn,
Arg, Met, Pro, Ser, Thr, or His”

Modified-site

/note= “Xaa at position 38 is Asn
or Gly”

Modified-site

/note= “Xaa at position 39 is Leu,
Met, or Phe”

Modified-site

/note= “Xaa at position 40 is Arg,
Ala, or Ser”

Modified-site

/note= “Xaa at position 41 is Arg,
Thr, Val, Leu, or Gly”

Modified-site

/note= “Xaa at position 42 is Pro,
Gly, Cys, Ser, Gln, Ala, Arg, Asn, Glu, His, Leu,
Thr, Val, or Lys”

Modified-site

/note= “Xaa at position 45 is Glu,
Tyr, His, Leu, or Arg”

Modified-site

/note= “Xaa at position 46 is Ala,
Ser, Asn, or Thr”

Modified-site

/note= “Xaa at position 47 is Phe
or Ser”

Modified-site

/note= “Xaa at position 48 is Asn,
Val, Pro, Thr, or Ile”

Modified-site

/note= “Xaa at position 49 is Arg,
Tyr, Lys, Ser, His, or Val”

Modified-site

/note= “Xaa at position 50 is Ala
or Asn”

Modified-site

/note= “Xaa at position 51 is Val,
Thr, Leu, or Ser”

Modified-site

/note= “Xaa at position 52 is Lys,
Ile, Arg, Val, Asn, Glu, or Ser”

Modified-site

/note= “Xaa at position 53 is Ser,
Phe, Val, Gly, Asn, Ile, or His”

Modified-site

/note= “Xaa at position 54 is Leu,
Val, Ile, Phe, or His”

Modified-site

/note= “Xaa at position 55 is Gln,
Ala, Pro, Thr, Glu, Arg, or Gly”

Modified-site

/note= “Xaa at position 56 is Asn
or Pro”

Modified-site

/note= “Xaa at position 57 is Ala,
Met, Pro, Arg, Glu, Thr, or Gln”

Modified-site

/note= “Xaa at position 58 is Ser,
Glu, Met, Ala, His, Asn, Arg, or Asp”

Modified-site

/note= “Xaa at position 59 is Ala,
Glu, Asp, Leu, Ser, Gly, Thr, Arg, or Pro”

Modified-site

/note= “Xaa at position 60 is Ile
or Met”

Modified-site

/note= “Xaa at position 61 is Glu,
Gly, Asp, Ser, or Gln”

Modified-site

/note= “Xaa at position 62 is Ser,
Val, Ala, Asn, Glu, Pro, Gly, or Asp”

Modified-site

/note= “Xaa at position 63 is Ile,
Ser, or Leu”

Modified-site

/note= “Xaa at position 65 is Lys,
Thr, Gly, Asn, Met, Arg, Ile, or Asp”

Modified-site

/note= “Xaa at position 66 is Asn,
Val, Gly, Thr, Leu, Glu, or Arg”

Modified-site

/note= “Xaa at position 67 is Leu
or Val”

Modified-site

/note= “Xaa at position 68 is Leu,
Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Met, Phe,
Ser, Thr, Tyr, or Val”

Modified-site

/note= “Xaa at position 69 is Pro,
Ala, Thr, Trp, or Met”

Modified-site

/note= “Xaa at position 71 is Leu
or Val”

Modified-site

/note= “Xaa at position 73 is Leu
or Ser”

Modified-site

/note= “Xaa at position 74 is Ala,
Arg, or Trp”

Modified-site

/note= “Xaa at position 75 is Thr,
Asp, Glu, His, Asn, or Ser”

Modified-site

/note= “Xaa at position 76 is Ala,
Asp, or Met”

Modified-site

/note= “Xaa at position 77 is Ala,
Pro, Ser, Thr, Phe, Leu, or Asp”

Modified-site

/note= “Xaa at position 78 is Pro
or Ser”

Modified-site

/note= “Xaa at position 79 is Thr,
Asp, Ser, Pro, Ala, Leu, or Arg”

Modified-site

/note= “Xaa at position 81 is His,
Pro, Arg, Val, Leu, Gly, Asn, Ile, Phe, Ser, or Thr”

Modified-site

/note= “Xaa at position 82 is Pro
or Tyr”

Modified-site

/note= “Xaa at position 83 is Ile,
Val, or Ala”

Modified-site

/note= “Xaa at position 84 is His,
Ile, Asn, Leu, Asp, Ala, Thr, Arg, Gln, Glu, Lys,
Met, Ser, Tyr, Val, or Pro”

Modified-site

/note= “Xaa at position 85 is Ile,
Leu, Val, or Phe”

Modified-site

/note= “Xaa at position 86 is Lys,
Leu, His, Arg, Ile, Gln, Pro, or Ser”

Modified-site

/note= “Xaa at position 87 is Asp,
Pro, Met, Lys, His, Thr, Val, Asn, Ile, Leu, or Tyr”

Modified-site

/note= “Xaa at position 88 is Gly,
Glu, Lys, or Ser”

Modified-site

/note= “Xaa at position 90 is Trp,
Val, Tyr, Met, or Leu”

Modified-site

/note=
“Xaa at position 91 is Asn, Pro, Ala, Phe, Ser,
Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His”

Modified-site

/note= “Xaa at position 92 is Glu,
Ser, Ala, or Gly”

Modified-site

/note= “Xaa at position 94 is Arg,
Ala, Gln, Ser, or Lys”

Modified-site

/note= “Xaa at position 95 Arg,
Thr, Glu, Leu, Ser, or Gly”

Modified-site

/note= “Xaa at position 98 is Thr,
Val, Gln, Glu, His, or Ser”

Modified-site

100

/note= “Xaa at position 100 is Tyr
or Trp”

Modified-site

101

/note= “Xaa at position 101 is Leu
or Ala”

Modified-site

102

/note= “Xaa at position 102 is Lys,
Thr, Met, Val, Trp, Ser, Leu, Ala, Asn, Gln, His,
Met, Phe, Tyr, or Ile”

Modified-site

103

/note= “Xaa at position 103 is Thr,
Ser, or Asn”

Modified-site

105

/note= “Xaa at position 105 is Glu,
Ser, Pro, Leu, Thr, or Tyr”

Modified-site

106

/note= “Xaa at position 106 is Asn,
Pro, Leu, His, Val, or Gln”

Modified-site

107

/note= “Xaa at position 107 is Ala,
Ser, Ile, Asn, Pro, Lys, Asp, or Gly”

Modified-site

108

/note=
“Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe,
Pro, His, Ile, Tyr, or Cys”

Modified-site

109

/note= “Xaa at position 109 is Ala,
Met, Glu, His, Ser, Pro, Tyr, or Leu”

20
Asn Cys Xaa Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Glu Xaa Xaa Xaa
20 25 30
Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Leu Xaa Xaa Xaa Xaa
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu
50 55 60
Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg
65 70 75 80
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Phe Xaa Xaa Lys
85 90 95
Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Gln Gln
100 105 110

111 amino acids

amino acid

linear

peptide

Modified-site

/note= “Met- or Met-Ala- may or may
not precede the amino acid in position 1”

Modified-site

/note= “Xaa at position 3 is Ser,
Gly, Asp, Met, or Gln”

Modified-site

/note= “Xaa at position 4 is Asn,
His, or Ile”

Modified-site

/note= “Xaa at position 5 is Met
or Ile”

Modified-site

/note= “Xaa at position 7 is Asp or
Glu”

Modified-site

/note= “Xaa at position 9 is Ile,
Ala, Leu, or Gly”

Modified-site

/note= “Xaa at position 10 is Ile,
Val, or Leu”

Modified-site

/note= “Xaa at position 11 is Thr,
His, Gln, or Ala”

Modified-site

/note= “Xaa at position 12 is His
or Ala”

Modified-site

/note= “Xaa at position 15 is Gln,
Asn, or Val”

Modified-site

/note= “Xaa at position 16 is Pro,
Gly, or Gln”

Modified-site

/note= “Xaa at position 17 is Pro,
Asp, Gly, or Gln”

Modified-site

/note= “Xaa at position 18 is Leu,
Arg, Gln, Asn, Gly, Ala, or Glu”

Modified-site

/note= “Xaa at position 19 is Pro
or Glu”

Modified-site

/note= “Xaa at position 20 is Leu,
Val, Gly, Ser, Lys, Ala, Arg, Gln, Glu, Ile, Phe,
Thr, or Met”

Modified-site

/note= “Xaa at position 21 is Leu,
Ala, Asn, Pro, Gln, or Val”

Modified-site

/note= “Xaa at position 23 is Phe,
Ser, Pro, or Trp”

Modified-site

/note= “Xaa at position 24 is Asn
or Ala”

Modified-site

/note= “Xaa at position 28 is Gly,
Asp, Ser, Cys, Ala, Asn, Ile, Leu, Met, Tyr, or Arg”

Modified-site

/note= “Xaa at position 30 is Asp
or Glu”

Modified-site

/note= “Xaa at position 31 is Gln,
Val, Met, Leu, Thr, Ala, Asn, Glu, Ser, or Lys”

Modified-site

/note= “Xaa at position 32 is Asp,
Phe, Ser, Thr, Ala, Asn, Gln, Glu, His, Ile, Lys,
Tyr, Val, or Cys”

Modified-site

/note= “Xaa at position 36 is Glu,
Ala, Asn, Ser, or Asp”

Modified-site

/note= “Xaa at position 37 is Asn,
Arg, Met, Pro, Ser, Thr, or His”

Modified-site

/note= “Xaa at position 40 is Arg
or Ala”

Modified-site

/note= “Xaa at position 41 is Arg,
Thr, Val, Leu, or Gly”

Modified-site

/note= “Xaa at position 42 is Pro,
Gly, Ser, Gln, Ala, Arg, Asn, Glu, Leu, Thr, Val,
or Lys”

Modified-site

/note= “Xaa at position 46 is Ala
or Ser”

Modified-site

/note= “Xaa at position 48 is Asn,
Pro, Thr, or Ile”

Modified-site

/note= “Xaa at position 49 is Arg
or Lys”

Modified-site

/note= “Xaa at position 50 is Ala
or Asn”

Modified-site

/note= “Xaa at position 51 is Val
or Thr”

Modified-site

/note= “Xaa at position 52 is Lys
or Arg”

Modified-site

/note= “Xaa at position 53 is Ser,
Phe, or His”

Modified-site

/note= “Xaa at position 54 is Leu,
Ile, Phe, or His”

Modified-site

/note= “Xaa at position 55 is Gln,
Ala, Pro, Thr, Glu, Arg, or Gly”

Modified-site

/note= “Xaa at position 57 is Ala,
Pro, or Arg”

Modified-site

/note= “Xaa at position 58 is Ser,
Glu, Arg, or Asp”

Modified-site

/note= “Xaa at position 59 is Ala
or Leu”

Modified-site

/note= “Xaa at position 62 is Ser,
Val, Ala, Asn, Glu, Pro, or Gly”

Modified-site

/note= “Xaa at position 63 is Ile
or Leu”

Modified-site

/note= “Xaa at position 65 is Lys,
Thr, Gly, Asn, Met, Arg, Ile, Gly, or Asp”

Modified-site

/note= “Xaa at position 66 is Asn,
Gly, Glu, or Arg”

Modified-site

/note= “Xaa at position 68 is Leu,
Gln, Trp, Arg, Asp, Ala, Asn, Glu, His, Ile, Met,
Phe, Ser, Thr, Tyr, or Val”

Modified-site

/note= “Xaa at position 69 is Pro
or Thr”

Modified-site

/note= “Xaa at position 71 is Leu
or Val”

Modified-site

/note= “Xaa at position 73 is Leu
or Ser”

Modified-site

/note= “Xaa at position 74 is Ala
or Trp”

Modified-site

/note= “Xaa at position 77 is Ala
or Pro”

Modified-site

/note= “Xaa at position 79 is Thr,
Asp, Ser, Pro, Ala, Leu, or Arg”

Modified-site

/note= “Xaa at position 81 is His,
Pro, Arg, Val, Leu, Gly, Asn, Phe, Ser, or Thr”

Modified-site

/note= “Xaa at position 82 is Pro
or Tyr”

Modified-site

/note= “Xaa at position 83 is Ile
or Val”

Modified-site

/note= “Xaa at position 84 is His,
Ile, Asn, Leu, Ala, Thr, Leu, Arg, Gln, Leu, Lys,
Met, Ser, Tyr, Val, or Pro”

Modified-site

/note= “Xaa at position 85 is Ile,
Leu, or Val”

Modified-site

/note= “Xaa at position 86 is Lys,
Arg, Ile, Gln, Pro, or Ser”

Modified-site

/note= “Xaa at position 87 is Asp,
Pro, Met, Lys, His, Thr, Asn, Ile, Leu, or Tyr”

Modified-site

/note= “Xaa at position 90 is Trp
or Leu”

Modified-site

/note=“Xaa at position 91 is Asn,
Pro, Ala, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp,
or His”

Modified-site

/note= “Xaa at position 92 is Glu
or Gly”

Modified-site

/note= “Xaa at position 94 is Arg,
Ala, or Ser”

Modified-site

/note= “Xaa at position 95 is Arg,
Thr, Glu, Leu, or Ser”

Modified-site

/note= “Xaa at position 98 is Thr,
Val, or Gln”

Modified-site

100

/note= “Xaa at position 100 is Tyr
or Trp”

Modified-site

101

/note= “Xaa at position 101 is Leu
or Ala”

Modified-site

102

/note= “Xaa at position 102 is Lys,
Thr, Val, Trp, Ser, Ala, His, Met, Phe, Tyr, or Ile”

Modified-site

103

/note= “Xaa at position 103 is Thr
or Ser”

Modified-site

106

/note= “Xaa at position 106 is Asn,
Pro, Leu, His, Val, or Gln”

Modified-site

107

/note= “Xaa at position 107 is Ala,
Ser, Ile, Asn, Pro, Asp, or Gly”

Modified-site

108

/note= “Xaa at position 108 is Gln,
Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys”

Modified-site

109

/note= “Xaa at position 109 is Ala,
Met, Glu, His, Ser, Pro, Tyr, or Leu”

21
Asn Cys Xaa Xaa Xaa Ile Xaa Glu Xaa Xaa Xaa Xaa Leu Lys Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Asn Leu Asn Xaa Glu Xaa Xaa Xaa
20 25 30
Ile Leu Met Xaa Xaa Asn Leu Xaa Xaa Xaa Asn Leu Glu Xaa Phe Xaa
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Ile Glu Xaa Xaa Leu
50 55 60
Xaa Xaa Leu Xaa Xaa Cys Xaa Pro Xaa Xaa Thr Ala Xaa Pro Xaa Arg
65 70 75 80
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Asp Xaa Xaa Xaa Phe Xaa Xaa Lys
85 90 95
Leu Xaa Phe Xaa Xaa Xaa Xaa Leu Glu Xaa Xaa Xaa Xaa Gln Gln
100 105 110

111 amino acids

amino acid

linear

peptide

Modified-site

/note= “Met- or Met-Ala- may or may
not precede the amino acid in position 1”

Modified-site

/note= “Xaa at position 3 is Ser,
Gly, Asp, or Gln”

Modified-site

/note= “Xaa at position 4 is Asn,
His, or Ile”

Modified-site

/note= “Xaa at position 9 is Ile,
Ala, Leu, or Gly”

Modified-site

/note= “Xaa at position 11 is Thr,
His, or Gln”

Modified-site

/note= “Xaa at position 12 is His
or Ala”

Modified-site

/note= “Xaa at position 15 is Gln
or Asn”

Modified-site

/note= “Xaa at position 16 is Pro
or Gly”

Modified-site

/note= “Xaa at position 18 is Leu,
Arg, Asn, or Ala”

Modified-site

/note= “Xaa at position 20 is Leu,
Val, Ser, Ala, Arg, Gln, Glu, Ile, Phe, Thr, or Met”

Modified-site

/note= “Xaa at position 21 is Leu,
Ala, Asn, or Pro”

Modified-site

/note= “Xaa at position 24 is Asn
or Ala”

Modified-site

/note= “Xaa at position 28 is Gly,
Asp, Ser, Ala, Asn, Ile, Leu, Met, Tyr, or Arg”

Modified-site

/note= “Xaa at position 31 is Gln,
Val, Met, Leu, Ala, Asn, Glu, or Lys”

Modified-site

/note= “Xaa at position 32 is Asp,
Phe, Ser, Ala, Gln, Glu, His, Val, or Thr”

Modified-site

/note= “Xaa at position 36 is Glu,
Asn, Ser, or Asp”

Modified-site

/note= “Xaa at position 37 is Asn,
Arg, Pro, Thr, or His”

Modified-site

/note= “Xaa at position 41 is Arg,
Leu, or Gly”

Modified-site

/note= “Xaa at position 42 is Pro,
Gly, Ser, Ala, Asn, Val, Leu, or Gln”

Modified-site

/note= “Xaa at position 48 is Asn,
Pro, or Thr”

Modified-site

/note= “Xaa at position 50 is Ala
or Asn”

Modified-site

/note= “Xaa at position 51 is Val
or Thr”

Modified-site

/note= “Xaa at position 53 is Ser
or Phe”

Modified-site

/note= “Xaa at position 54 is Leu
or Phe”

Modified-site

/note= “Xaa at position 55 is Gln,
Ala, Glu, or Arg”

Modified-site

/note= “Xaa at position 62 is Ser,
Val, Asn, Pro, or Gly”

Modified-site

/note= “Xaa at position 63 is Ile
or Leu”

Modified-site

/note= “Xaa at position 65 is Lys,
Asn, Met, Arg, Ile, or Gly”

Modified-site

/note= “Xaa at position 66 is Asn,
Gly, Glu, or Arg”

Modified-site

/note= “Xaa at position 68 is Leu,
Gln, Trp, Arg, Asp, Asn, Glu, His, Met, Phe, Ser,
Thr, Tyr, or Val”

Modified-site

/note= “Xaa at position 73 is Leu
or Ser”

Modified-site

/note= “Xaa at position 74 is Ala
or Trp”

Modified-site

/note= “Xaa at position 77 is Ala
or Pro”

Modified-site

/note= “Xaa at position 79 is Thr,
Asp, or Ala”

Modified-site

/note= “Xaa at position 81 is His,
Pro, Arg, Val, Gly, Asn, Ser, or Thr”

Modified-site

/note= “Xaa at position 84 is His,
Ile, Asn, Ala, Thr, Arg, Gln, Glu, Lys, Met,
Ser, Tyr, Val, or Leu”

Modified-site

/note= “Xaa at position 85 is Ile
or Leu”

Modified-site

/note= “Xaa at position 86 is Lys
or Arg”

Modified-site

/note= “Xaa at position 87 is Asp,
Pro, Met, Lys, His, Pro, Asn, Ile, Leu, or Tyr”

Modified-site

/note= “Xaa at position 91 is Asn,
Pro, Ser, Ile, or Asp”

Modified-site

/note= “Xaa at position 94 is Arg,
Ala, or Ser”

Modified-site

/note= “Xaa at position 95 is Arg,
Thr, Glu, Leu, or Ser”

Modified-site

/note= “Xaa at position 98 is Thr
or Gln”

Modified-site

102

/note= “Xaa at position 102 is Lys,
Val, Trp, or Ile”

Modified-site

103

/note= “Xaa at position 103 is Thr,
Ala, His, Phe, Tyr, or Ser”

Modified-site

106

/note= “Xaa at position 106 is Asn,
Pro, Leu, His, Val, or Gln”

Modified-site

107

/note= “Xaa at position 107 is Ala,
Ser, Ile, Pro, or Asp”

Modified-site

108

/note= “Xaa at position 108 is Gln,
Met, Trp, Phe, Pro, His, Ile, or Tyr”

Modified-site

109

/note= “Xaa at position 109 is Ala,
Met, Glu, Ser, or Leu”

22
Asn Cys Xaa Xaa Met Ile Asp Glu Xaa Ile Xaa Xaa Leu Lys Xaa Xaa
1 5 10 15
Pro Xaa Pro Xaa Xaa Asp Phe Xaa Asn Leu Asn Xaa Glu Asp Xaa Xaa
20 25 30
Ile Leu Met Xaa Xaa Asn Leu Arg Xaa Xaa Asn Leu Glu Ala Phe Xaa
35 40 45
Arg Xaa Xaa Lys Xaa Xaa Xaa Asn Ala Ser Ala Ile Glu Xaa Xaa Leu
50 55 60
Xaa Xaa Leu Xaa Pro Cys Leu Pro Xaa Xaa Thr Ala Xaa Pro Xaa Arg
65 70 75 80
Xaa Pro Ile Xaa Xaa Xaa Xaa Gly Asp Trp Xaa Glu Phe Xaa Xaa Lys
85 90 95
Leu Xaa Phe Tyr Leu Xaa Xaa Leu Glu Xaa Xaa Xaa Xaa Gln Gln
100 105 110

111 amino acids

amino acid

linear

peptide

Modified-site

/note= “Met- or Met-Ala- may or may
not precede the amino acid in position 1”

23
Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro
1 5 10 15
Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp
20 25 30
Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn
35 40 45
Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu
50 55 60
Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg
65 70 75 80
His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys
85 90 95
Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

24
CTAGCCACGG CCGCACCCAC GCGACATCCA ATCCATATCA AGGACGGTGA CTGGAATG 58

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

25
TTAACATTCC AGTCACCGTC CTTGATATGG ATTGGATGTC GCGTGGGTGC GGCCGTGG 58

20 base pairs

nucleic acid

single

linear

DNA (synthetic)

26
TGTCTGCTCA GGCCATGGCT 20

67 base pairs

nucleic acid

single

linear

DNA (synthetic)

27
GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGTCGAC TTATTACGTG GGTGCGGCCG 60
TGGCTAG 67

55 base pairs

nucleic acid

single

linear

DNA (synthetic)

28
GCGCGAATTC ATTCCAGTCA CCGTCGACTT ATTAGATTGG ATGTCGCGTG GGTGC 55

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

29
TGAACCATAT GTCAGG 16

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

30
AATTCCTGAC ATATGGTTCA TGCA 24

43 base pairs

nucleic acid

single

linear

DNA (synthetic)

31
GCGCGAATTC GTCGACTTAT TAGTCCTTGA TATGGATTGG ATG 43

67 base pairs

nucleic acid

single

linear

DNA (synthetic)

32
GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATTGG ATGSNNCGTG GGTGCGGCCG 60
TGGCTAG 67

64 base pairs

nucleic acid

single

linear

DNA (synthetic)

33
GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATTGG SNNTCGCGTG GGTGCGGCCG 60
TGGC 64

61 base pairs

nucleic acid

single

linear

DNA (synthetic)

34
GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGGATSNN ATGTCGCGTG GGTGCGGCCG 60
T 61

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

35
GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TATGSNNTGG ATGTCGCGTG GGTGCGGC 58

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

36
GATATGGATT GGATGTCGCG TGGG 24

55 base pairs

nucleic acid

single

linear

DNA (synthetic)

37
GCGCGAATTC ATTCCAGTCA CCGTCCTTGA TSNNGATTGG ATGTCGCGTG GGTGC 55

52 base pairs

nucleic acid

single

linear

DNA (synthetic)

38
GCGCGAATTC ATTCCAGTCA CCGTCCTTSN NATGGATTGG ATGTCGCGTG GG 52

49 base pairs

nucleic acid

single

linear

DNA (synthetic)

39
GCGCGAATTC ATTCCAGTCA CCGTCSNNGA TATGGATTGG ATGTCGCGT 49

46 base pairs

nucleic acid

single

linear

DNA (synthetic)

40
GCGCGAATTC ATTCCAGTCA CCSNNCTTGA TATGGATTGG ATGTCG 46

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

41
GTCACCGTCC TTGATATGGA TTGG 24

43 base pairs

nucleic acid

single

linear

DNA (synthetic)

42
GCGCGAATTC ATTCCAGTCS NNGTCCTTGA TATGGATTGG ATG 43

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

43
GCGCGAATTC ATTCCASNNA CCGTCCTTGA TATGGATTGG 40

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

44
GCGCGAATTC ATTSNNGTCA CCGTCCTTGA TATGGAT 37

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

45
GCGCGAATTC SNNCCAGTCA CCGTCCTTGA TATG 34

25 base pairs

nucleic acid

single

linear

DNA (synthetic)

46
GAATTCATTC CAGTCACCGT TCCTT 25

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

47
CGCGCGGAAT TCATTCCAGT CACCGT 26

42 base pairs

nucleic acid

single

linear

DNA (synthetic)

48
CGCGCGCCAT GGCTAACTGC ATTATAACAC ACACTTAAAG CA 42

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

49
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAACA GCCACCTTTG CCTTTGCT 58

76 base pairs

nucleic acid

single

linear

DNA (synthetic)

50
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCTGG 60
ACTTCAACAA CCTCAA 76

50 base pairs

nucleic acid

single

linear

DNA (synthetic)

51
GCGCGCGATA TCTTGGTCTT CTTCACCATT CAGCGGCAGC GGTGGCTGCT 50

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

52
GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATGAGGT TGTTGAAGTC 60
CAGCA 65

47 base pairs

nucleic acid

single

linear

DNA (synthetic)

53
GCGCGCCTCG AGGTTTGGAC GACGAAGATC TTGGTCTTCA CCATTGA 47

68 base pairs

nucleic acid

single

linear

DNA (synthetic)

54
GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGT TATTTTCCAT 60
CAGGATAT 68

50 base pairs

nucleic acid

single

linear

DNA (synthetic)

55
GCGCGCTGAT GCATTCTGCA GAGACTTGAC GAGGTTTGGA CGACGAAGGT 50

47 base pairs

nucleic acid

single

linear

DNA (synthetic)

56
GCGCGCCTCG AGGCATTCAA CCGTGCTGCA TCAGCAATTG AGAGCAT 47

35 base pairs

nucleic acid

single

linear

DNA (synthetic)

57
GCGCGCCTGC AGAATATTCT TAAAAATCTC CTGCC 35

53 base pairs

nucleic acid

single

linear

DNA (synthetic)

58
GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCCCATGTC TGCCGCTAGC CAC 53

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

59
GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GACGGCCGCA 60
CCCACGCGAC A 71

67 base pairs

nucleic acid

single

linear

DNA (synthetic)

60
CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCA GGGCAGACAT 60
GGCAGGA 67

78 base pairs

nucleic acid

single

linear

DNA (synthetic)

61
CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTAT 60
TCCAGTCACC GTCCTTGA 78

60 base pairs

nucleic acid

single

linear

DNA (synthetic)

62
CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA CAGTTTACGA CGGAATTCAT 60

42 base pairs

nucleic acid

single

linear

DNA (synthetic)

63
CGCGCGAAGC TTATTACTGT TGGGTTTTCA GATAGAAGGT CA 42

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

64
CGCGCGCCAT GGCTAACTGC NNSAACATGA TCGATGAAAT TATAACACAC TTAAAGCA 58

111 amino acids

amino acid

linear

peptide

65
Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro
1 5 10 15
Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp
20 25 30
Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn
35 40 45
Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu
50 55 60
Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg
65 70 75 80
His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys
85 90 95
Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110

113 amino acids

amino acid

linear

peptide

66
Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys
1 5 10 15
Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp
20 25 30
Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala
35 40 45
Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser
50 55 60
Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro
65 70 75 80
Thr Arg His Pro Ile Ile Ile Arg Asp Gly Asp Trp Asn Glu Phe Arg
85 90 95
Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln
100 105 110
Gln

113 amino acids

amino acid

linear

peptide

67
Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys
1 5 10 15
Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp
20 25 30
Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala
35 40 45
Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser
50 55 60
Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro
65 70 75 80
Thr Arg Arg Pro Ile Ile Ile Arg Asp Gly Asp Trp Asn Glu Phe Arg
85 90 95
Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln
100 105 110
Gln

339 base pairs

nucleic acid

double

linear

DNA (genomic)

68
ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA GCCACCGCTG 60
CCGCTGCTGG ACTTCAACAA CCTCAATGAC GAAGACATGT CTATCCTGAT GGAAAATAAC 120
CTTCGTCGTC CAAACCTCGA GGCATTCAAC CGTGCTGTCA AGTCTCTGCA GAATGCATCA 180
GCAATTGAGA GCATTCTTAA AAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC 240
ACGCGACATC CAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACC 300
TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAG 339

113 amino acids

amino acid

linear

peptide

69
Met Ala Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys
1 5 10 15
Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Asp Glu Asp
20 25 30
Met Ser Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala
35 40 45
Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser
50 55 60
Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro
65 70 75 80
Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg
85 90 95
Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln
100 105 110
Gln

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

70
CTTTAAGTGT GTTATAATTT CGTTGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

71
CATGGCTAAC TGCTCTAACA TGATCCAAGA AATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

72
CTTTAAGTGT GTTATAATTT CTTGGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

73
CATGGCTAAC TGCTCTAACA TGATCGAAGA AATTATAACA 40

76 base pairs

nucleic acid

single

linear

DNA (synthetic)

74
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACANNS TTAAAGCAGC 60
CACCTTTGCC TTTGCT 76

76 base pairs

nucleic acid

single

linear

DNA (synthetic)

75
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC NNSAAGCAGC 60
CACCTTTGCC TTTGCT 76

76 base pairs

nucleic acid

single

linear

DNA (synthetic)

76
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTANNSCAGC 60
CACCTTTGCC TTTGCT 76

94 base pairs

nucleic acid

single

linear

DNA (synthetic)

77
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGNNSC 60
CACCTTTGCC TTTGCTGGAC TTCAACAACC TCAA 94

94 base pairs

nucleic acid

single

linear

DNA (synthetic)

78
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGN 60
NSCCTTTGCC TTTGCTGGAC TTCAACAACC TCAA 94

94 base pairs

nucleic acid

single

linear

DNA (synthetic)

79
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC 60
CANNSTTGCC TTTGCTGGAC TTCAACAACC TCAA 94

94 base pairs

nucleic acid

single

linear

DNA (synthetic)

80
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC 60
CACCTNNSCC TTTGCTGGAC TTCAACAACC TCAA 94

94 base pairs

nucleic acid

single

linear

DNA (synthetic)

81
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC 60
CACCTTTGNN STTGCTGGAC TTCAACAACC TCAA 94

94 base pairs

nucleic acid

single

linear

DNA (synthetic)

82
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATAACACAC TTAAAGCAGC 60
CACCTTTGCC TNNSCTGGAC TTCAACAACC TCAA 94

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

83
GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTGAAG TCSNNCAGCG GCAGCGGTGG 60
CTGCT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

84
GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTGAAS NNCAGCAGCG GCAGCGGTGG 60
CTGCT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

85
GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTGTTSNNG TCCAGCAGCG GCAGCGGTGG 60
CTGCT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

86
GCGCGCGATA TCTTGGTCTT CACCATTGAG GTTSNNGAAG TCCAGCAGCG GCAGCGGTGG 60
CTGCT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

87
GCGCGCGATA TCTTGGTCTT CACCATTGAG SNNGTTGAAG TCCAGCAGCG GCAGCGGTGG 60
CTGCT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

88
GCGCGCGATA TCTTGGTCTT CACCATTSNN GTTGTTGAAG TCCAGCAGCG GCAGCGGTGG 60
CTGCT 65

83 base pairs

nucleic acid

single

linear

DNA (synthetic)

89
GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCTTCACC 60
SNNGAGGTTG TTGAAGTCCA GCA 83

83 base pairs

nucleic acid

single

linear

DNA (synthetic)

90
GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCTTCSNN 60
ATTGAGGTTG TTGAAGTCCA GCA 83

83 base pairs

nucleic acid

single

linear

DNA (synthetic)

91
GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GGTCSNNACC 60
ATTGAGGTTG TTGAAGTCCA GCA 83

83 base pairs

nucleic acid

single

linear

DNA (synthetic)

92
GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCTT GSNNTTCACC 60
ATTGAGGTTG TTGAAGTCCA GCA 83

83 base pairs

nucleic acid

single

linear

DNA (synthetic)

93
GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATATCSN NGTCTTCACC 60
ATTGAGGTTG TTGAAGTCCA GCA 83

83 base pairs

nucleic acid

single

linear

DNA (synthetic)

94
GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGGATSNNTT GGTCTTCACC 60
ATTGAGGTTG TTGAAGTCCA GCA 83

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

95
GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATC AGSNNATCTT GGTCTTCACC 60
ATTGA 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

96
GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCCATS NNGATATCTT GGTCTTCACC 60
ATTGA 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

97
GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTTTCSNNC AGGATATCTT GGTCTTCACC 60
ATTGA 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

98
GCGCGCCTCG AGGTTTGGAC GACGAAGGTT ATTSNNCATC AGGATATCTT GGTCTTCACC 60
ATTGA 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

99
GCGCGCCTCG AGGTTTGGAC GACGAAGGTT SNNTTCCATC AGGATATCTT GGTCTTCACC 60
ATTGA 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

100
GCGCGCCTCG AGGTTTGGAC GACGAAGSNN ATTTTCCATC AGGATATCTT GGTCTTCACC 60
ATTGA 65

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

101
GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGACG 60
ACGSNNGTTA TTTTCCATCA GGATAT 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

102
GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGACG 60
SNNAAGGTTA TTTTCCATCA GGATAT 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

103
GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTTGGSNN 60
ACGAAGGTTA TTTTCCATCA GGATAT 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

104
GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GGTTSNNACG 60
ACGAAGGTTA TTTTCCATCA GGATAT 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

105
GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCGA GSNNTGGACG 60
ACGAAGGTTA TTTTCCATCA GGATAT 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

106
GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCCTCSN NGTTTGGACG 60
ACGAAGGTTA TTTTCCATCA GGATAT 86

68 base pairs

nucleic acid

single

linear

DNA (synthetic)

107
GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AATGCSNNGA GGTTTGGACG 60
ACGAAGGT 68

68 base pairs

nucleic acid

single

linear

DNA (synthetic)

108
GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTG AASNNCTCGA GGTTTGGACG 60
ACGAAGGT 68

68 base pairs

nucleic acid

single

linear

DNA (synthetic)

109
GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGGTTS NNTGCCTCGA GGTTTGGACG 60
ACGAAGGT 68

68 base pairs

nucleic acid

single

linear

DNA (synthetic)

110
GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCACGSNNG AATGCCTCGA GGTTTGGACG 60
ACGAAGGT 68

68 base pairs

nucleic acid

single

linear

DNA (synthetic)

111
GCGCGCTGAT GCATTCTGCA GAGACTTGAC AGCSNNGTTG AATGCCTCGA GGTTTGGACG 60
ACGAAGGT 68

68 base pairs

nucleic acid

single

linear

DNA (synthetic)

112
GCGCGCTGAT GCATTCTGCA GAGACTTGAC SNNACGGTTG AATGCCTCGA GGTTTGGACG 60
ACGAAGGT 68

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

113
GCGCGCCTCG AGGCATTCAA CCGTGCTNNS AAGTCTCTGC AGAATGCATC AGCAATTGAG 60
AGCAT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

114
GCGCGCCTCG AGGCATTCAA CCGTGCTGTC NNSTCTCTGC AGAATGCATC AGCAATTGAG 60
AGCAT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

115
GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGNNSCTGC AGAATGCATC AGCAATTGAG 60
AGCAT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

116
GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTNNSC AGAATGCATC AGCAATTGAG 60
AGCAT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

117
GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTCTGN NSAATGCATC AGCAATTGAG 60
AGCAT 65

65 base pairs

nucleic acid

single

linear

DNA (synthetic)

118
GCGCGCCTCG AGGCATTCAA CCGTGCTGTC AAGTCTCTGC AGNNSGCATC AGCAATTGAG 60
AGCAT 65

53 base pairs

nucleic acid

single

linear

DNA (synthetic)

119
GCGCGCCTGC AGAATNNSTC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCC 53

53 base pairs

nucleic acid

single

linear

DNA (synthetic)

120
GCGCGCCTGC AGAATGCANN SGCAATTGAG AGCATTCTTA AAAATCTCCT GCC 53

53 base pairs

nucleic acid

single

linear

DNA (synthetic)

121
GCGCGCCTGC AGAATGCATC ANNSATTGAG AGCATTCTTA AAAATCTCCT GCC 53

53 base pairs

nucleic acid

single

linear

DNA (synthetic)

122
GCGCGCCTGC AGAATGCATC AGCANNSGAG AGCATTCTTA AAAATCTCCT GCC 53

53 base pairs

nucleic acid

single

linear

DNA (synthetic)

123
GCGCGCCTGC AGAATGCATC AGCAATTNNS AGCATTCTTA AAAATCTCCT GCC 53

53 base pairs

nucleic acid

single

linear

DNA (synthetic)

124
GCGCGCCTGC AGAATGCATC AGCAATTGAG NNSATTCTTA AAAATCTCCT GCC 53

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

125
GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCNNSCTTA AAAATCTCCT GCCATGTCTG 60
CCGCTAGCCA C 71

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

126
GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTNNSA AAAATCTCCT GCCATGTCTG 60
CCGCTAGCCA C 71

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

127
GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTN NSAATCTCCT GCCATGTCTG 60
CCGCTAGCCA C 71

134 amino acids

amino acid

linear

peptide

128
Met Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn
1 5 10 15
Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro
20 25 30
Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile
35 40 45
Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg
50 55 60
Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys
65 70 75 80
Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His
85 90 95
Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu
100 105 110
Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr
115 120 125
Leu Ser Leu Ala Ile Phe
130

133 amino acids

amino acid

linear

peptide

Modified-site

/note= “Met- may or may not precede
the amino acid in position 1”

Modified-site

/note= “Xaa at position 18 is Asn
or Ile”

Modified-site

/note= “Xaa at position 25 is Thr
or His”

Modified-site

/note= “Xaa at position 29 is Gln,
Arg, or Val”

Modified-site

/note= “Xaa at position 32 is Leu,
Ala, or Asn”

Modified-site

/note= “Xaa at position 37 is Phe,
Pro, or Ser”

Modified-site

/note= “Xaa at position 42 is Glu,
Ala, or Ser”

Modified-site

/note= “Xaa at position 45 is Gln,
Val, or Met”

Modified-site

/note= “Xaa at position 51 is Asn
or Arg”

Modified-site

/note= “Xaa at position 55 is Arg,
Leu, or Thr”

Modified-site

/note= “Xaa at position 59 is Glu
or Leu”

Modified-site

/note= “Xaa at position 60 is Ala
or Ser”

Modified-site

/note= “Xaa at position 62 is Asn
or Val”

Modified-site

/note= “Xaa at position 67 is Ser,
Asn, or His”

Modified-site

/note= “Xaa at position 69 is Gln
or Glu”

Modified-site

/note= “Xaa at position 73 is Ala
or Gly”

Modified-site

/note= “Xaa at position 76 is Ser
or Ala”

Modified-site

/note= “Xaa at position 79 is Lys
or Arg”

Modified-site

/note= “Xaa at position 82 is Leu,
Glu, or Val”

Modified-site

/note= “Xaa at position 87 is Leu
or Ser”

Modified-site

/note= “Xaa at position 93 is Pro
or Ser”

Modified-site

/note= “Xaa at position 98 is His,
Ile, or Thr”

Modified-site

101

/note= “Xaa at position 101 is Asp
or Ala”

Modified-site

105

/note= “Xaa at position 105 is Asn
or Glu”

Modified-site

109

/note= “Xaa at position 109 is Arg
or Glu”

Modified-site

116

/note= “Xaa at position 116 is Lys
or Val”

Modified-site

120

/note= “Xaa at position 120 is Asn,
Gln, or His”

Modified-site

123

/note= “Xaa at position 123 is Ala
or Glu”

129
Ala Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys
1 5 10 15
Ser Xaa Met Ile Asp Glu Ile Ile Xaa His Leu Lys Xaa Pro Pro Xaa
20 25 30
Pro Leu Leu Asp Xaa Asn Asn Leu Asn Xaa Glu Asp Xaa Asp Ile Leu
35 40 45
Met Glu Xaa Asn Leu Arg Xaa Pro Asn Leu Xaa Xaa Phe Xaa Arg Ala
50 55 60
Val Lys Xaa Leu Xaa Asn Ala Ser Xaa Ile Glu Xaa Ile Leu Xaa Asn
65 70 75 80
Leu Xaa Pro Cys Leu Pro Xaa Ala Thr Ala Ala Pro Xaa Arg His Pro
85 90 95
Ile Xaa Ile Lys Xaa Gly Asp Trp Xaa Glu Phe Arg Xaa Lys Leu Thr
100 105 110
Phe Tyr Leu Xaa Thr Leu Glu Xaa Ala Gln Xaa Gln Gln Thr Thr Leu
115 120 125
Ser Leu Ala Ile Phe
130

111 amino acids

amino acid

linear

peptide

Modified-site

/note= “Met- or Met-Ala may or may
not precede the amino acid in position 1”

Modified-site

/note= “Xaa at position 4 is Asn or
Ile”

Modified-site

/note= “Xaa at position 11 is Thr
or His”

Modified-site

/note= “Xaa at position 15 is Gln,
Arg, or Val”

Modified-site

/note= “Xaa at position 18 is Leu,
Ala, or Asn”

Modified-site

/note= “Xaa at position 23 is Phe,
Pro, or Ser”

Modified-site

/note= “Xaa at position 28 is Glu,
Ala, or Ser”

Modified-site

/note= “Xaa at position 31 is Gln,
Val, or Met”

Modified-site

/note= “Xaa at position 37 is Asn
or Arg”

Modified-site

/note= “Xaa at position 41 is Arg,
Leu, or Thr”

Modified-site

/note= “Xaa at position 45 is Glu
or Leu”

Modified-site

/note= “Xaa at position 46 is Ala
or Ser”

Modified-site

/note= “Xaa at position 48 is Asn
or Val”

Modified-site

/note= “Xaa at position 53 is Ser,
Asn, or His”

Modified-site

/note= “Xaa at position 55 is Gln
or Glu”

Modified-site

/note= “Xaa at position 59 is Ala
or Gly”

Modified-site

/note= “Xaa at position 62 is Ser
or Ala”

Modified-site

/note= “Xaa at position 65 is Lys
or Arg”

Modified-site

/note= “Xaa at position 68 is Leu,
Glu, or Val”

Modified-site

/note= “Xaa at position 73 is Leu
or Ser”

Modified-site

/note= “Xaa at position 79 is Pro
or Ser”

Modified-site

/note= “Xaa at position 84 is His,
Ile, or Thr”

Modified-site

/note= “Xaa at position 87 is Asp
or Ala”

Modified-site

/note= “Xaa at position 91 is Asn
or Glu”

Modified-site

/note= “Xaa at position 95 is Arg
or Glu”

Modified-site

102

/note= “Xaa at position 102 is Lys
or Val”

Modified-site

106

/note= “Xaa at position 106 is Asn,
Gln, or His”

Modified-site

109

/note= “Xaa at position 109 is Ala
or Glu”

130
Asn Cys Ser Xaa Met Ile Asp Glu Ile Ile Xaa His Leu Lys Xaa Pro
1 5 10 15
Pro Xaa Pro Leu Leu Asp Xaa Asn Asn Leu Asn Xaa Glu Asp Xaa Asp
20 25 30
Ile Leu Met Glu Xaa Asn Leu Arg Xaa Pro Asn Leu Xaa Xaa Phe Xaa
35 40 45
Arg Ala Val Lys Xaa Leu Xaa Asn Ala Ser Xaa Ile Glu Xaa Ile Leu
50 55 60
Xaa Asn Leu Xaa Pro Cys Leu Pro Xaa Ala Thr Ala Ala Pro Xaa Arg
65 70 75 80
His Pro Ile Xaa Ile Lys Xaa Gly Asp Trp Xaa Glu Phe Arg Xaa Lys
85 90 95
Leu Thr Phe Tyr Leu Xaa Thr Leu Glu Xaa Ala Gln Xaa Gln Gln
100 105 110

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

131
CTAGCCACGG CCGCACCCAC GCGACATCCA ATCCATATCA AGGACGGTGA CTGGAATG 58

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

132
TTAACATTCC AGTCACCGTC CTTGATATGG ATTGGATGTC GCGTGGGTGC GGCCGTGG 58

29 base pairs

nucleic acid

double

linear

DNA (genomic)

133
AAGGAGATAT ATCCATGAAC TGCTCTAAC 29

5 amino acids

amino acid

linear

peptide

134
Met Asn Cys Ser Asn
1 5

120 amino acids

amino acid

linear

peptide

135
Met Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln
1 5 10 15
Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln
20 25 30
Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe
35 40 45
Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile
50 55 60
Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr
65 70 75 80
Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg
85 90 95
Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110
Thr Thr Leu Arg Leu Ala Ile Phe
115 120

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

136
AATTCCGTCG TAAACTGACC TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAGA 60
CCACTCTGTC G 71

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

137
CTAGCGACAG AGTGGTCTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTCA 60
GTTTACGACG G 71

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

138
GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AANNSCTCCT GCCATGTCTG 60
CCGCTAGCCA C 71

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

139
GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATNNSCT GCCATGTCTG 60
CCGCTAGCCA C 71

71 base pairs

nucleic acid

single

linear

DNA (synthetic)

140
GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCNN SCCATGTCTG 60
CCGCTAGCCA C 71

89 base pairs

nucleic acid

single

linear

DNA (synthetic)

141
GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GNNSTGTCTG 60
CCGCTAGCCA CGGCCGCACC CACGCGACA 89

89 base pairs

nucleic acid

single

linear

DNA (synthetic)

142
GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCANNSCTG 60
CCGCTAGCCA CGGCCGCACC CACGCGACA 89

89 base pairs

nucleic acid

single

linear

DNA (synthetic)

143
GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTNNS 60
CCGCTAGCCA CGGCCGCACC CACGCGACA 89

408 base pairs

nucleic acid

double

linear

DNA (genomic)

144
ATGGCTCCAA TGACTCAGAC TACTTCTCTT AAGACTTCTT GGGTTAACTG CTCTAACATG 60
ATCGATGAAA TTATAACACA CTTAAAGCAG CCACCTTTGC CTTTGCTGGA CTTCAACAAC 120
CTCAATGGGG AAGACCAAGA CATTCTGATG GAAAATAACC TTCGAAGGCC AAACCTGGAG 180
GCATTCAACA GGGCTGTCAA GAGTTTACAG AATGCATCAG CAATTGAGAG CATTCTTAAA 240
AATCTCCTGC CATGTCTGCC CCTGGCCACG GCCGCACCCA CGCGACATCC AATCCATATC 300
AAGGACGGTG ACTGGAATGA ATTCCGTCGT AAACTGACCT TCTATCTGAA AACCTTGGAG 360
AACGCGCAGG CTCAACAGAC CACTCTGTCG CTAGCGATCT TTTAATAA 408

157 base pairs

nucleic acid

double

linear

DNA (genomic)

CDS

1..156

145
ATC GAT GAA ATC ATC ACC CAC CTG AAG CAG CCA CCG CTG CCG CTG CTG 48
Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu
1 5 10 15
GAC TTC AAC AAC CTC AAT GGT GAA GAC CAA GAT ATC CTG ATG GAA AAT 96
Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn
20 25 30
AAC CTT CGT CGT CCA AAC CTC GAG GCA TTC AAC CGT GCT GTC AAG TCT 144
Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser
35 40 45
CTG CAG AAT GCA T 157
Leu Gln Asn Ala
50

52 amino acids

amino acid

linear

protein

146
Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu
1 5 10 15
Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn
20 25 30
Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser
35 40 45
Leu Gln Asn Ala
50

414 base pairs

nucleic acid

double

linear

DNA (genomic)

147
CCATGGCTCC AATGACTCAG ACTACTTCTC TTAAGACTTC TTGGGTTAAC TGCTCTAACA 60
TGATCGATGA AATTATAACA CACTTAAAGC AGCCACCTTT GCCTTTGCTG GACTTCAACA 120
ACCTCAATGG GGAAGACCAA GACATTCTGA TGGAAAATAA CCTTCGAAGG CCAAACCTGG 180
AGGCATTCAA CAGGGCTGTC AAGAGTTTAC AGAATGCATC AGCAATTGAG AGCATTCTTA 240
AAAATCTCCT GCCATGTCTG CCCCTGGCCA CGGCCGCACC CACGCGACAT CCAATCCATA 300
TCAAGGACGG TGACTGGAAT GAATTCCGTC GTAAACTGAC CTTCTATCTG AAAACCTTGG 360
AGAACGCGCA GGCTCAACAG ACCACTCTGT CGCTAGCGAT CTTTTAATAA GCTT 414

414 base pairs

nucleic acid

double

linear

DNA (genomic)

148
AAGCTTATTA AAAGATCGCT AGCGACAGAG TGGTCTGTTG AGCCTGCGCG TTCTCCAAGG 60
TTTTCAGATA GAAGGTCAGT TTACGACGGA ATTCATTCCA GTCACCGTCC TTGATATGGA 120
TTGGATGTCG CGTGGGTGCG GCCGTGGCCA GGGGCAGACA TGGCAGGAGA TTTTTAAGAA 180
TGCTCTCAAT TGCTGATGCA TTCTGTAAAC TCTTGACAGC CCTGTTGAAT GCCTCCAGGT 240
TTGGCCTTCG AAGGTTATTT TCCATCAGAA TGTCTTGGTC TTCCCCATTG AGGTTGTTGA 300
AGTCCAGCAA AGGCAAAGGT GGCTGCTTTA AGTGTGTTAT AATTTCATCG ATCATGTTAG 360
AGCAGTTAAC CCAAGAAGTC TTAAGAGAAG TAGTCTGAGT CATTGGAGCC ATGG 414

81 base pairs

nucleic acid

double

linear

DNA (genomic)

149
ATGATGATTA CTCTGCGCAA ACTTCCTCTG GCGGTTGCCG TCGCAGCGGG CGTAATGTCT 60
GCTCAGGCCA TGGCTAACTG C 81

81 base pairs

nucleic acid

double

linear

DNA (genomic)

150
GCAGTTAGCC ATGGCCTGAG CAGACATTAC GCCCGCTGCG ACGGCAACCG CCAGAGGAAG 60
TTTGCGCAGA GTAATCATCA T 81

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

151
CATGGCTAAC TGCTCTAACA TGAT 24

22 base pairs

nucleic acid

single

linear

DNA (synthetic)

152
CGATCATGTT AGAGCAGTTA GC 22

12 base pairs

nucleic acid

double

linear

DNA (genomic)

153
ATGGCTAACT GC 12

4 amino acids

amino acid

linear

peptide

154
Met Ala Asn Cys
1

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

155
GCCGATACCG CGGCATACTC CCACCATTCA GAGA 34

33 base pairs

nucleic acid

single

linear

DNA (synthetic)

156
GCCGATAAGA TCTAAAACGG GTATGGAGAA ACA 33

89 base pairs

nucleic acid

single

linear

DNA (synthetic)

157
GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG 60
NNSCTAGCCA CGGCCGCACC CACGCGACA 89

89 base pairs

nucleic acid

single

linear

DNA (synthetic)

158
GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG 60
CCGNNSGCCA CGGCCGCACC CACGCGACA 89

89 base pairs

nucleic acid

single

linear

DNA (synthetic)

159
GCGCGCCTGC AGAATGCATC AGCAATTGAG AGCATTCTTA AAAATCTCCT GCCATGTCTG 60
CCGCTANNSA CGGCCGCACC CACGCGACA 89

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

160
CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGTGCGGC 60
SNNGGCCAGG GGCAGACATG GCAGGA 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

161
CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGTGCSNN 60
CGTGGCCAGG GGCAGACATG GCAGGA 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

162
CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TGGGSNNGGC 60
CGTGGCCAGG GGCAGACATG GCAGGA 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

163
CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGCG TSNNTGCGGC 60
CGTGGCCAGG GGCAGACATG GCAGGA 86

86 base pairs

nucleic acid

single

linear

DNA (synthetic)

164
CGCGCGGAAT TCATTCCAGT CACCGTCCTT GATATGGATT GGATGTCGSN NGGGTGCGGC 60
CGTGGCCAGG GGCAGACATG GCAGGA 86

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

165
TTTCAGATAG AAGGTCAGTT TACGACGGAA SNNATTCCAG TCACCGTC 48

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

166
TTTCAGATAG AAGGTCAGTT TACGACGSNN TTCATTCCAG TCACCGTC 48

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

167
TTTCAGATAG AAGGTCAGTT TACGSNNGAA TTCATTCCAG TCACCGTC 48

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

168
TTTCAGATAG AAGGTCAGTT TSNNACGGAA TTCATTCCAG TCACCGTC 48

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

169
TTTCAGATAG AAGGTCAGSN NACGACGGAA TTCATTCCAG TCACCGTC 48

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

170
TTTCAGATAG AAGGTSNNTT TACGACGGAA TTCATTCCAG TCACCGTC 48

23 base pairs

nucleic acid

single

linear

DNA (synthetic)

171
CGCGCGAAGC TTATTACTGT TGA 23

78 base pairs

nucleic acid

single

linear

DNA (synthetic)

172
CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TAGAASNNCA 60
GTTTACGACG GAATTCAT 78

78 base pairs

nucleic acid

single

linear

DNA (synthetic)

173
CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGA TASNNGGTCA 60
GTTTACGACG GAATTCAT 78

78 base pairs

nucleic acid

single

linear

DNA (synthetic)

174
CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTCAGS NNGAAGGTCA 60
GTTTACGACG GAATTCAT 78

78 base pairs

nucleic acid

single

linear

DNA (synthetic)

175
CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTTTTSNNA TAGAAGGTCA 60
GTTTACGACG GAATTCAT 78

78 base pairs

nucleic acid

single

linear

DNA (synthetic)

176
CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA GGTSNNCAGA TAGAAGGTCA 60
GTTTACGACG GAATTCAT 78

78 base pairs

nucleic acid

single

linear

DNA (synthetic)

177
CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCCAA SNNTTTCAGA TAGAAGGTCA 60
GTTTACGACG GAATTCAT 78

60 base pairs

nucleic acid

single

linear

DNA (synthetic)

178
CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTCTCSNN GGTTTTCAGA TAGAAGGTCA 60

60 base pairs

nucleic acid

single

linear

DNA (synthetic)

179
CGCGCGAAGC TTATTACTGT TGAGCCTGCG CGTTSNNCAA GGTTTTCAGA TAGAAGGTCA 60

60 base pairs

nucleic acid

single

linear

DNA (synthetic)

180
CGCGCGAAGC TTATTACTGT TGAGCCTGCG CSNNCTCCAA GGTTTTCAGA TAGAAGGTCA 60

60 base pairs

nucleic acid

single

linear

DNA (synthetic)

181
CGCGCGAAGC TTATTACTGT TGAGCCTGSN NGTTCTCCAA GGTTTTCAGA TAGAAGGTCA 60

60 base pairs

nucleic acid

single

linear

DNA (synthetic)

182
CGCGCGAAGC TTATTACTGT TGAGCSNNCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTCA 60

60 base pairs

nucleic acid

single

linear

DNA (synthetic)

183
CGCGCGAAGC TTATTACTGT TGSNNCTGCG CGTTCTCCAA GGTTTTCAGA TAGAAGGTCA 60

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

184
TGCTCTAACA TGATCGATGA AATT 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

185
GAAATTATAA CACACTTAAA GCAG 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

186
AAGCAGCCAC CTTTGCCTTT GCTG 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

187
AAGCAGCCAC CGCTGCCGCT GCTG 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

188
CTCAATGGTG AAGACCAAGA TATC 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

189
GATATCCTGA TGGAAAATAA CCTT 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

190
AACCTTCGTC GTCCAAACCT CGAG 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

191
CTCGAGGCAT TCAACCGTGC TGTC 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

192
GCTGTCAAGT CTCTGCAGAA TGCA 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

193
AATGCATCAG CAATTGAGAG CATT 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

194
AGCATTCTTA AAAATCTCCT GCCA 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

195
CTGCCATGTC TGCCCCTGGC CACG 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

196
CTGGCCACGG CCGCACCCAC GCGA 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

197
AATGAATTCC GTCGTAAACT GACC 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

198
CTGACCTTCT ATCTGAAAAC CTTG 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

199
ACCTTGGAGA ACGCGCAGGC TCAA 24

22 base pairs

nucleic acid

single

linear

DNA (synthetic)

200
GAATGCATCA GCAATTGAGA GC 22

20 base pairs

nucleic acid

single

linear

DNA (synthetic)

201
AATTGCTGAT GCATTCTGCA 20

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

202
ATTCTTAAAA ATCTCCTGCC ATGT 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

203
CAGGAGATTT TTAAGAATGC TCTC 24

30 base pairs

nucleic acid

single

linear

DNA (synthetic)

204
CTGCCCCTGG CCACGGCCGC ACCCACGCGA 30

30 base pairs

nucleic acid

single

linear

DNA (synthetic)

205
GGGTGCGGCC GTGGCCAGGG GCAGACATGG 30

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

206
CATCCAATCA TCATCCGTGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

207
AATTCATTCC AGTCACCGTC ACGGATGATG ATTGGATGTC GCGT 44

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

208
CGCCCAATCA TCATCCGTGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

209
AATTCATTCC AGTCACCGTC ACGGATGATG ATTGGGCGTC GCGT 44

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

210
CATGGCTAAC TGCTCTAACA TGATCGATGA AATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

211
CTTTAAGTGT GTTATAATTT CATCGATCAT GTTAGAGCAG TTAGC 45

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

212
CACTTAAAGC AGCCACCTTT GCCTTTGCTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

213
GAGGTTGTTG AAGTCCAGCA AAGGCAAAGG TGGCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

214
AACAACCTCA ATGACGAAGA CATGTCT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

215
AGACATGTCT TCGTCATT 18

339 base pairs

nucleic acid

double

linear

DNA (genomic)

216
ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA GCCACCGCTG 60
CCGCTGCTGG ACTTCAACAA CCTCAATGGT GAAGACCAAG ATATCCTGAT GGAAAATAAC 120
CTTCGTCGTC CAAACCTCGA GGCATTCAAC CGTGCTGTCA ACTCTCTGCA GAATGCATCA 180
GCAATTGAGA GCATTCTTAA AAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC 240
ACGCGACATC CAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACC 300
TTCTATCTGA AAACCTTGGA GAACGCGCAG GCTCAACAG 339

111 amino acids

amino acid

linear

peptide

217
Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro
1 5 10 15
Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp
20 25 30
Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn
35 40 45
Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu
50 55 60
Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg
65 70 75 80
His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys
85 90 95
Leu Thr Phe Tyr Leu Trp Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110

111 amino acids

amino acid

linear

peptide

218
Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro
1 5 10 15
Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp
20 25 30
Ile Leu Met Asp Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn
35 40 45
Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu
50 55 60
Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg
65 70 75 80
His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys
85 90 95
Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln
100 105 110

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

219
CTTTAAGTGT GTTATAATTT CTTCGATCAT GTTAGAGCAG TTAGC 45

339 base pairs

nucleic acid

double

linear

DNA (genomic)

220
ATGGCTAACT GCTCTAACAT GATCGATGAA ATCATCACCC ACCTGAAGCA GCCACCGCTG 60
CCGCTGCTGG ACTTCAACAA CCTCAATGGT GAAGACCAAG ATATCCTGAT GGAAAATAAC 120
CTTCGTCGTC CAAACCTCGA GGCATTCAAC CGTGCTGTCA ACTCTCTGCA GAATGCATCA 180
GCAATTGAGA GCATTCTTAA AAATCTCCTG CCATGTCTGC CCCTGGCCAC GGCCGCACCC 240
ACGCGACATC CAATCCATAT CAAGGACGGT GACTGGAATG AATTCCGTCG TAAACTGACC 300
TTCTATCTGT GGACCTTGGA GAACGCGCAG GCTCAACAG 339

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

221
CATGGCTAAC TGCTCTAACA TGATCAACGA AATTATAACA 40

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

222
CGCGCGCCAT GGCTAACTGC TCTNNSATGA TCGATGAAAT TATAACACAC TTAAAGCA 58

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

223
CGCGCGCCAT GGCTAACTGC TCTAACNNSA TCGATGAAAT TATAACACAC TTAAAGCA 58

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

224
CGCGCGCCAT GGCTAACTGC TCTAACATGN NSGATGAAAT TATAACACAC TTAAAGCA 58

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

225
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCNNSGAAAT TATAACACAC TTAAAGCA 58

58 base pairs

nucleic acid

single

linear

DNA (synthetic)

226
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATNNSAT TATAACACAC TTAAAGCA 58

76 base pairs

nucleic acid

single

linear

DNA (synthetic)

227
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAANN SATAACACAC TTAAAGCAGC 60
CACCTTTGCC TTTGCT 76

76 base pairs

nucleic acid

single

linear

DNA (synthetic)

228
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TNNSACACAC TTAAAGCAGC 60
CACCTTTGCC TTTGCT 76

76 base pairs

nucleic acid

single

linear

DNA (synthetic)

229
CGCGCGCCAT GGCTAACTGC TCTAACATGA TCGATGAAAT TATANNSCAC TTAAAGCAGC 60
CACCTTTGCC TTTGCT 76

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

230
CATGGCTAAC TGCTCTAACA TGATCAGCGA AATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

231
CTTTAAGTGT GTTATAATTT CGCTGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

232
CATGGCTAAC TGCTCTAACA TGATCACCGA AATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

233
CTTTAAGTGT GTTATAATTT CCGTGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

234
CATGGCTAAC TGCTCTAACA TGATCGATAA CATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

235
CTTTAAGTGT GTTATAATGT TATCGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

236
CATGGCTAAC TGCTCTAACA TGATCGATGA CATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

237
CTTTAAGTGT GTTATAATGT CATCGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

238
CATGGCTAAC TGCTCTAACA TGATCGATCA GATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

239
CTTTAAGTGT GTTATAATCT GATCGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

240
CATGGCTAAC TGCTCTAACA TGATCGATCT GATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

241
CTTTAAGTGT GTTATAATCA GATCGATCAT GTTAGAGCAG TTAGC 45

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

242
CATGGCTAAC TGCTCTAACA TGATCGATGT TATTATAACA 40

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

243
CTTTAAGTGT GTTATAATAA CATCGATCAT GTTAGAGCAG TTAGC 45

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

244
CACTTAAAGC AGCCACCTTT GCCTGCTCTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

245
GAGGTTGTTG AAGTCCAGAG CAGGCAAAGG TGGCTG 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

246
CACTTAAAGC AGCCACCTTT GCCTCGTCTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

247
GAGGTTGTTG AAGTCCAGAC GAGGCAAAGG TGGCTG 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

248
CACTTAAAGC AGCCACCTTT GCCTCAGCTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

249
GAGGTTGTTG AAGTCCAGCT GAGGCAAAGG TGGCTG 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

250
CACTTAAAGC AGCCACCTTT GCCTGAACTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

251
GAGGTTGTTG AAGTCCAGCT CAGGCAAAGG TGGCTG 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

252
CACTTAAAGC AGCCACCTTT GCCTATCCTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

253
GAGGTTGTTG AAGTCCAGGA TAGGCAAAGG TGGCTG 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

254
CACTTAAAGC AGCCACCTTT CCCTTTCCTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

255
GAGGTTGTTG AAGTCCAGGA AAGGCAAAGG TGGCTG 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

256
CACTTAAAGC AGCCACCTTT GCCTACCCTG GACTTC 36

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

257
GAGGTTGTTG AAGTCCAGGG TAGGCAAAGG TGGCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

258
AACAACCTCA ATCGTGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

259
ATCTTGGTCT TCACGATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

260
AACAACCTCA ATAACGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

261
ATCTTGGTCT TCGTTATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

262
AACAACCTCA ATGAAGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

263
ATCTTGGTCT TCTTCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

264
AACAACCTCA ATATCGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

265
ATCTTGGTCT TCGATATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

266
AACAACCTCA ATCTGGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

267
ATCTTGGTCT TCCAGATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

268
AACAACCTCA ATAAAGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

269
ATCTTGGTCT TCTTTATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

270
AACAACCTCA ATATGGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

271
ATCTTGGTCT TCCATATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

272
AACAACCTCA ATTTCGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

273
ATCTTGGTCT TCGAAATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

274
AACAACCTCA ATACCGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

275
ATCTTGGTCT TCGGTATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

276
AACAACCTCA ATTACGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

277
ATCTTGGTCT TCGTAATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

278
AACAACCTCA ATGTTGAAGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

279
ATCTTGGTCT TCAACATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

280
AACAACCTCA ATGGGCGTGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

281
ATCTTGGTCT CGCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

282
AACAACCTCA ATGGGCAGGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

283
ATCTTGGTCC TGCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

284
AACAACCTCA ATGGGGGTGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

285
ATCTTGGTCA CCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

286
AACAACCTCA ATGGGACCGA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

287
ATCTTGGTCG GTCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

288
AACAACCTCA ATGGGGAAGC TCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

289
ATCTTGAGCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

290
AACAACCTCA ATGGGGAAAA CCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

291
ATCTTGGTTT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

292
AACAACCTCA ATGGGGAACA GCAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

293
ATCTTGCTGT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

294
AACAACCTCA ATGGGGAAGA ACAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

295
ATCTTGTTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

296
AACAACCTCA ATGGGGAAGA CGCTGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

297
ATCAGCGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

298
AACAACCTCA ATGGGGAAGA CCGTGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

299
ATCACGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

300
AACAACCTCA ATGGGGAAGA CAACGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

301
ATCGTTGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

302
AACAACCTCA ATGGGGAAGA CGACGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

303
ATCGTCGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

304
AACAACCTCA ATGGTGAAGA CGAAGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

305
ATCTTCGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

306
AACAACCTCA ATGGTGAAGA CCACGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

307
ATCGTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

308
AACAACCTCA ATGGGGAAGA CATCGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

309
ATCGATGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

310
AACAACCTCA ATGGGGAAGA CTCCGAT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

311
ATCGGAGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

312
AACAACCTCA ATGGGGAAGA CCAAGCT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

313
AGCTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

314
AACAACCTCA ATGGGGAAGA CCAAAAC 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

315
GTTTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

316
AACAACCTCA ATGGGGAAGA CCAACAG 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

317
CTGTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

318
AACAACCTCA ATGGGGAAGA CCAAGAA 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

319
TTCTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

320
AACAACCTCA ATGGGGAAGA CCAACAC 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

321
GTGTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

322
AACAACCTCA ATGGGGAAGA CCAAATC 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

323
GATTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

324
AACAACCTCA ATGGGGAAGA CCAACTG 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

325
CAGTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

326
AACAACCTCA ATGGGGAAGA CCAAAAA 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

327
TTTTTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

328
AACAACCTCA ATGGGGAAGA CCAATAC 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

329
GTATTGGTCT TCCCCATT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

330
AACAACCTCA ATGGGGAAGA CCAAGTT 27

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

331
AACTTGGTCT TCCCCATT 18

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

332
ATCGCTATGG AAAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

333
CCTTCGAAGG TTATTTTCCA TAGCGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

334
ATCGAAATGG AAAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

335
CCTTCGAAGG TTATTTTCCA TTTCGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

336
ATCAAAATGG AAAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

337
CCTTCGAAGG TTATTTTCCA TTTTGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

338
ATCATGATGG AAAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

339
CCTTCGAAGG TTATTTTCCA TCATGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

340
ATCACCATGG AAAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

341
CCTTCGAAGG TTATTTTCCA TGGTGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

342
ATCGTTATGG AAAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

343
CCTTCGAAGG TTATTTTCCA TAACGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

344
ATCCTGATGC ACAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

345
CCTTCGAAGG TTATTGTGCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

346
ATCCTGATGA TGAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

347
CCTTCGAAGG TTATTCATCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

348
ATCCTGATGT TCAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

349
CCTTCGAAGG TTATTGAACA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

350
ATCCTGATGG CTAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

351
CCTTCGAAGG TTATTAGCCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

352
ATCCTGATGA ACAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

353
CCTTCGAAGG TTATTGTTCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

354
ATCCTGATGA TCAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

355
CCTTCGAAGG TTATTGATCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

356
ATCCTGATGA AAAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

357
CCTTCGAAGG TTATTTTTCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

358
ATCCTGATGT CCAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

359
CCTTCGAAGG TTATTGGACA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

360
ATCCTGATGG TTAATAACCT TCGAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

361
CCTTCGAAGG TTATTAACCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

362
ATCCTGATGG AAAATAACCT TGCTAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

363
CCTAGCAAGG TTATTTTCCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

364
ATCCTGATGG AAAATAACCT TAACAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

365
CCTGTTAAGG TTATTTTCCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

366
ATCCTGATGG AAAATAACCT TCACAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

367
CCTGTGAAGG TTATTTTCCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

368
ATCCTGATGG AAAATAACCT TAAAAGGCCA AACCTG 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

369
CCTTTTAAGG TTATTTTCCA TCAGGAT 27

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

370
ATCCTGATGG AAAATAACCT TCGAAGGGCT AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

371
CCTGTTGAAT GCCTCCAGGT TAGC 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

372
ATCCTGATGG AAAATAACCT TCGAAGGCGT AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

373
CCTGTTGAAT GCCTCCAGGT TACG 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

374
ATCCTGATGG AAAATAACCT TCGAAGGAAC AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

375
CCTGTTGAAT GCCTCCAGGT TGTT 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

376
ATCCTGATGG AAAATAACCT TCGAAGGGAA AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

377
CCTGTTGAAT GCCTCCAGGT TTTC 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

378
ATCCTGATGG AAAATAACCT TCGAAGGCAC AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

379
CCTGTTGAAT GCCTCCAGGT TGTG 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

380
ATCCTGATGG AAAATAACCT TCGAAGGCTG AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

381
CCTGTTGAAT GCCTCCAGGT TCAG 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

382
ATCCTGATGG AAAATAACCT TCGAAGGTTC AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

383
CCTGTTGAAT GCCTCCAGGT TGAA 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

384
ATCCTGATGG AAAATAACCT TCGAAGGACC AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

385
CCTGTTGAAT GCCTCCAGGT TGGT 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

386
ATCCTGATGG AAAATAACCT TCGAAGGTAC AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

387
CCTGTTGAAT GCCTCCAGGT TGTA 24

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

388
ATCCTGATGG AAAATAACCT TCGAAGGGTT AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

389
CCTGTTGAAT GCCTCCAGGT TAAC 24

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

390
AAAAATCTCG CTCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

391
AGCGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

392
AAAAATCTCA ACCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

393
GTTGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

394
AAAAATCTCG AACCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

395
TTCGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

396
AAAAATCTCC ACCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

397
GTGGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

398
AAAAATCTCA TCCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

399
GATGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

400
AAAAATCTCA TGCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

401
CATGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

402
AAAAATCTCT TCCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

403
GAAGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

404
AAAAATCTCT CCCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

405
GGAGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

406
AAAAATCTCA CCCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

407
GGTGAGATTT TTAAGAAT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

408
AAAAATCTCT ACCCATGT 18

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

409
GTAGAGATTT TTAAGAAT 18

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

410
CTGCCCCTGG CCACGGCCGC AGCTACG 27

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

411
ATGGATTGGA TGTCGCGTAG CTGC 24

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

412
CTGCCCCTGG CCACGGCCGC AGGTACG 27

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

413
ATGGATTGGA TGTCGCGTAC CTGC 24

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

414
CTGCCCCTGG CCACGGCCGC AATCACG 27

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

415
ATGGATTGGA TGTCGCGTGA TTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

416
GCTCATCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

417
ATGGATTGGA TGAGCCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

418
CAGCATCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

419
ATGGATTGGA TGCTGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

420
CACCATCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

421
ATGGATTGGA TGGTGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

422
AAACATCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

423
ATGGATTGGA TGTTTCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

424
CGAGCTCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

425
ATGGATTGGA GCTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

426
CGAAACCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

427
ATGGATTGGG TTTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

428
CGAGACCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

429
ATGGATTGGG TCTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

430
CGAATCCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

431
ATGGATTGGG ATTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

432
CGAAAACCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

433
ATGGATTGGT TTTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

434
CGAATGCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

435
ATGGATTGGC ATTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

436
CGATTCCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

437
ATGGATTGGG AATCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

438
CGATCCCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

439
ATGGATTGGG GATCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

440
CGATGGCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

441
ATGGATTGGC CATCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

442
CGATACCCAA TCCATATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

443
ATGGATTGGG TATCGCGTGG GTGC 24

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

444
CATCCAATCC AAATCAAGGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

445
AATTCATTCC AGTCACCGTC CTTGATTTGG ATTGGATGTC GCGT 44

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

446
CATCCAATCG AAATCAAGGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

447
AATTCATTCC AGTCACCGTC CTTGATTTCG ATTGGATGTC GCGT 44

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

448
CATCCAATCA TGATCAAGGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

449
AATTCATTCC AGTCACCGTC CTTGATCATG ATTGGATGTC GCGT 44

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

450
CATCCAATCT TCATCAAGGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

451
AATTCATTCC AGTCACCGTC CTTGATGAAG ATTGGATGTC GCGT 44

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

452
CATCCAATCT CCATCAAGGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

453
AATTCATTCC AGTCACCGTC CTTGATGGAG ATTGGATGTC GCGT 44

34 base pairs

nucleic acid

single

linear

DNA (synthetic)

454
CATCCAATCG TAATCAAGGA CGGTGACTGG AATG 34

44 base pairs

nucleic acid

single

linear

DNA (synthetic)

455
AATTCATTCC AGTCACCGTC CTTGATTACG ATTGGATGTC GCGT 44

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

456
CGACATCCAA TCCGTATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

457
ACGGATTGGA TGTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

458
CGACATCCAA TCAAAATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

459
TTTGATTGGA TGTCGCGTGG GTGC 24

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

460
CGACATCCAA TCTACATCAA G 21

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

461
GTAGATTGGA TGTCGCGTGG GTGC 24

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

462
GCTGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

463
AATTCATTCC AGTCACCAGC CTTGAT 26

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

464
AACGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

465
AATTCATTCC AGTCACCGTT CTTGAT 26

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

466
GAAGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

467
AATTCATTCC AGTCACCTTC CTTGAT 26

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

468
GGTGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

469
AATTCATTCC AGTCACCACC CTTGAT 26

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

470
ATCGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

471
AATTCATTCC AGTCACCGAT CTTGAT 26

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

472
CTGGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

473
AATTCATTCC AGTCACCCAG CTTGAT 26

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

474
TTCGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

475
AATTCATTCC AGTCACCGAA CTTGAT 26

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

476
TCCGGTGACT GGAATG 16

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

477
AATTCATTCC AGTCACCGGA CTTGAT 26

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

478
AATTCGCTAG GAAACTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

479
CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTAGCG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

480
AATTCCAGAG GAAACTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

481
CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTCTGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

482
AATTCCACAG GAAACTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

483
CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTGTGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

484
AATTCTCCAG GAAACTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

485
CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTGGAG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

486
AATTCCGGAG GCGTCTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

487
CTCAAGGGTT TTCAGATAGA ACGTCAGACG CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

488
AATTCCGGAG GGAACTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

489
CTCAAGGGTT TTCAGATAGA ACGTCAGTTC CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

490
AATTCCGGAG GCACCTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

491
CTCAAGGGTT TTCAGATAGA ACGTCAGGTG CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

492
AATTCCGGAG GATCCTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

493
CTCAAGGGTT TTCAGATAGA ACGTCAGGAT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

494
AATTCCGGAG GTCCCTGACG TTCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

495
CTCAAGGGTT TTCAGATAGA ACGTCAGGGA CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

496
AATTCCGGAG GAAACTGACG GACTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

497
CTCAAGGGTT TTCAGATAGT CCGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

498
AATTCCGGAG GAAACTGACG ATCTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

499
CTCAAGGGTT TTCAGATAGA TCGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

500
AATTCCGGAG GAAACTGACG CTGTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

501
CTCAAGGGTT TTCAGATACA GCGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

502
AATTCCGGAG GAAACTGACG AAATATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

503
CTCAAGGGTT TTCAGATATT TCGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

504
AATTCCGGAG GAAACTGACG GTTTATCTGA AA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

505
CTCAAGGGTT TTCAGATAAA CCGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

506
AATTCCGGAG GAAACTGACG TTCTATCTGG CT 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

507
CTCAAGGGTA GCCAGATAGA ACGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

508
AATTCCGGAG GAAACTGACG TTCTATCTGC GT 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

509
CTCAAGGGTA CGCAGATAGA ACGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

510
AATTCCGGAG GAAACTGACG TTCTATCTGA AC 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

511
CTCAAGGGTG TTCAGATAGA ACGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

512
AATTCCGGAG GAAACTGACG TTCTATCTGC AG 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

513
CTCAAGGGTC TGCAGATAGA ACGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

514
AATTCCGGAG GAAACTGACG TTCTATCTGC AC 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

515
CTCAAGGGTG TGCAGATAGA ACGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

516
AATTCCGGAG GAAACTGACG TTCTATCTGA TG 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

517
CTCAAGGGTC ATCAGATAGA ACGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

518
AATTCCGGAG GAAACTGACG TTCTATCTGT TC 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

519
CTCAAGGGTG AACAGATAGA ACGTCAGTTT CCTCCGG 37

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

520
AATTCCGGAG GAAACTGACG TTCTATCTGT AC 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

521
CTCAAGGGTG TACAGATAGA ACGTCAGTTT CCTCCGG 37

40 base pairs

nucleic acid

single

linear

DNA (synthetic)

522
CATGGCTAAC TGCTCTAACA TGATCGATGA AATTATAACA 40

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

523
CACTTAAAGC AGCCACCTTT GCCTTTGCTG GACTTC 36

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

524
AACAACCTCA ATGGGGAAGA CCAAGAT 27

45 base pairs

nucleic acid

single

linear

DNA (synthetic)

525
CTTTAAGTGT GTTATAATTT CATCGATCAT GTTAGAGCAG TTAGC 45

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

526
GAGGTTGTTG AAGTCCAGCA AAGGCAAAGG TGGCTG 36

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

527
ATCTTGGTCT TCCCCATT 18

36 base pairs

nucleic acid

single

linear

DNA (synthetic)

528
ATCCTGATGG AAAATAACCT TCGAAGGCCA AACCTG 36

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

529
GAGGCATTCA ACAGGGCTGT CAAG 24

15 base pairs

nucleic acid

single

linear

DNA (synthetic)

530
AGTTTACAGA ATGCA 15

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

531
CCTTCGAAGG TTATTTTCCA TCAGGAT 27

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

532
CCTGTTGAAT GCCTCCAGGT TTGG 24

20 base pairs

nucleic acid

single

linear

DNA (synthetic)

533
TTCTGTAAAC TCTTGACAGC 20

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

534
TCAGCAATTG AGAGCATTCT T 21

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

535
AAAAATCTCC TGCCATGT 18

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

536
CTGCCCCTGG CCACGGCCGC ACCCACGCGA CATCCAATCC ATATCAAG 48

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

537
CTGCCCCTGG CCACGGCCGC ACCCACG 27

21 base pairs

nucleic acid

single

linear

DNA (synthetic)

538
CGACATCCAA TCCATATCAA G 21

16 base pairs

nucleic acid

single

linear

DNA (synthetic)

539
GACGGTGACT GGAATG 16

19 base pairs

nucleic acid

single

linear

DNA (synthetic)

540
GCTCTCAATT GCTGATGCA 19

18 base pairs

nucleic acid

single

linear

DNA (synthetic)

541
CAGGAGATTT TTAAGAAT 18

48 base pairs

nucleic acid

single

linear

DNA (synthetic)

542
ATGGATTGGA TGTCGCGTGG GTGCGGCCGT GGCCAGGGGC AGACATGG 48

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

543
GGCCGTGGCC AGGGGCAGAC ATGG 24

24 base pairs

nucleic acid

single

linear

DNA (synthetic)

544
ATGGATTGGA TGTCGCGTGG GTGC 24

26 base pairs

nucleic acid

single

linear

DNA (synthetic)

545
AATTCATTCC AGTCACCGTC CTTGAT 26

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

546
AATTCCGGAG GAAACTGACG TTCTATCTGA AA 32

32 base pairs

nucleic acid

single

linear

DNA (synthetic)

547
ACCCTTGAGA ATGCGCAGGC TCAACAGTAA TA 32

37 base pairs

nucleic acid

single

linear

DNA (synthetic)

548
CTCAAGGGTT TTCAGATAGA ACGTCAGTTT CCTCCGG 37

27 base pairs

nucleic acid

single

linear

DNA (synthetic)

549
AGCTTATTAC TGTTGAGCCT GCGCATT 27

Claims

1. A method for selective ex-vivo expansion of stem cells, comprising the steps of;(a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:15; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); whereinXaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly; Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 36 is Asp, Leu, or Val; Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 38 is Asn, or Ala; Xaa at position 40 is Leu, Trp, or Arg; Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro; Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala; Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His; Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 57 is Asn or Gly; Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg; Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp; Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 85 is Leu, Asn, Val, or Gln; Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 87 is Leu, Ser, Trp, or Gly; Xaa at position 88 is Ala, Lys, Arg, Val, or Trp; Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro; Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr; Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 97 is Ile, Val, Lys, Ala, or Asn; Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 101 is Asp; Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 103 is Asp, or Ser; Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro; Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp; Xaa at position 111 is Leu, Ile, Arg, Asp, or Met; Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 116 is Lys; Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus; and(c) harvesting said cultured cells.
2. A method for selective ex-vivo expansion of stem cells, comprising the steps of,(a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:19; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); whereinXaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly; Xaa at position 9 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or, Ala; Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 22 is Asp, Leu, or Val; Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 24 is Asn, or Ala; Xaa at position 26 is Leu, Trp, or Arg; Xaa at position 27 is Asn, Cyg, Arg, Leu, His, Met, Pro; Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, Phe, Tyr, Ile or Met; Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 30 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, Ser, Ala, Ile, Glu, His or Trp; Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 33 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 34 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 43 is Asn or Gly; Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 45 is Glu Tyr, His, Leu, Pro, or Arg; Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 48 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 49 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp; Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 71 is Leu, Asn, Val, or Gln; Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 73 is Leu, Ser, Trp, or Gly; Xaa at position 74 is Ala, Lys, Arg, Val, or Trp; Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 79 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala or Pro; Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile or Tyr; Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 83 is Ile, Val, Lys, Ala, or Asn; Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 87 is Asp; Xaa at position 88 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 89 is Asp, or Ser; Xaa at position 90 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro; Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala or Trp; Xaa at position 97 is Leu, Ile, Arg, Asp, or Met; Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 102 is Lys; Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding native amino acids of (1-133) human interleukin-3; and(c) harvesting said cultured cells.
3. A method for selective ex-vivo expansion of stem cells, comprising the steps of;(a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting (i) the sequence of SEQ ID NO:129; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein; Xaa at position 18 is Asn or Ile; Xaa at position 19 is Met, Ala or Ile; Xaa at position 20 is Ile, Pro or Leu; Xaa at position 23 is Ile, Ala or Leu; Xaa at position 25 is Thr or His; Xaa at position 29 is Gln, Arg, Val or Ile; Xaa at position 32 is Leu, Ala, Asn or Arg; Xaa at position 34 is Leu or Ser; Xaa at position 37 is Phe, Pro, or Ser; Xaa at position 38 is Asn or Ala; Xaa at position 42 is Gly, Ala, Ser, Asp or Asn; Xaa at position 45 is Gln, Val, or Met; Xaa at position 46 is Asp or Ser; Xaa at position 49 is Met, Ile, Leu or Asp; Xaa at position 50 is Glu or Asp; Xaa at position 51 is Asn Arg or Ser; Xaa at position 55 is Arg, Leu, or Thr; Xaa at position 56 is Pro or Ser; Xaa at position 59 is Glu or Leu; Xaa at position 60 is Ala or Ser; Xaa at position 62 is Asn, Val or Pro; Xaa at position 63 is Arg or His; Xaa at position 65 is Val or Ser; Xaa at position 67 is Ser, Asn, His or Gln; Xaa at position 69 is Gln or Glu; Xaa at position 73 is Ala or Gly; Xaa at position 76 is Ser, Ala or Pro; Xaa at position 79 is Lys, Arg or Ser; Xaa at position 82 is Leu, Glu, Val or Trp; Xaa at position 85 is Leu or Val; Xaa at position 87 is Leu, Ser, Tyr; Xaa at position 88 is Ala or Trp; Xaa at position 91 is Ala or Pro; Xaa at position 93 is Pro or Ser; Xaa at position 95 is His or Thr; Xaa at position 98 is His, Ile, or Thr; Xaa at position 100 is Lys or Arg; Xaa at position 101 is Asp, Ala; Xaa at position 105 is Asn or Glu; Xaa at position 109 is Arg, Glu or Leu; Xaa at position 112 is Thr or Gln; Xaa at position 116 is Lys, Val, Xaa at position 117 is Thr or Ser; Xaa at position 120 is Asn, Gln, or His; Xaa at position 123 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3; and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus; and(c) harvesting said cultured cells.
4. A method for selective ex-vivo expansion of stem cells, comprising the steps of;(a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:130; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 4 is Asn or Ile; Xaa at position 5 is Met, Ala or Ile: Xaa at position 6 is Ile, Pro or Leu; Xaa at position 9 is Ile, Ala or Leu; Xaa at position 11 is Thr or His; Xaa at position 15 is Gln, Arg, Val or Ile; Xaa at position 18 is Leu, Ala, Asn or Arg; Xaa at position 20 is Leu or Ser; Xaa at position 23 is Phe, Pro, or Ser; Xaa at position 24 is Asn or Ala; Xaa at position 28 is Gly, Ala, Ser, Asp or Asn; Xaa at position 31 is Gln, Val, or Met; Xaa at position 32 is Asp or Ser; Xaa at position 35 is Met, Ile or Asp; Xaa at position 36 is Glu or Asp; Xaa at position 37 is Asn, Arg or Ser; Xaa at position 41 is Arg, Leu, or Thr; Xaa at position 42 is Pro or Ser; Xaa at position 45 is Glu or Leu; Xaa at position 46 is Ala or Ser; Xaa at position 48 is Asn, Val or Pro; Xaa at position 49 is Arg or His, Xaa at position 51 is Val or Ser; Xaa at position 53 is Ser, Asn, His or Gln; Xaa at position 55 is Gln or Glu; Xaa at position 59 is Ala or Gly; Xaa at position 62 is Ser, Ala or Pro; Xaa at position 65 is Lys, Arg or Ser; Xaa at position 67 is Leu, Glu, or Val; Xaa at position 68 is Leu, Glu, Val or Trp; Xaa at position 71 is Leu or Val; Xaa at position 73 is Leu, Ser or Tyr; Xaa at position 74 is Ala or Trp; Xaa at position 77 is Ala or Pro; Xaa at position 79 is Pro or Ser; Xaa at position 81 is His or Thr; Xaa at position 84 is His, Ile, or Thr; Xaa at position 86 is Lys or Arg; Xaa at position 87 is Asp, Ala; Xaa at position 91 is Asn or Glu; Xaa at position 95 is Arg, Glu Leu; Xaa at position 98 Thr or Gln; Xaa at position 102 is Lys, Val, Xaa at position 103 is Thr or Ser; Xaa at position 106 is Asn, Gln, or His; Xaa at position 109 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3; and(c) harvesting said cultured cells.
5. A method for the treatment of a patient having a hematopoietic disorder, comprising the steps of;(a) removing stem cells from said patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:15; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); whereinXaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly; Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 36 is Asp, Leu, or Val; Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 38 is Asn, or Ala; Xaa at position 40 is Leu, Trp, or Arg; Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro; Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala; Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His; Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 57 is Asn or Gly; Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg; Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp; Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 85 is Leu, Asn, Val, or Gln; Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 87 is Leu, Ser, Trp, or Gly; Xaa at position 88 is Ala, Lys, Arg, Val, or Trp; Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro; Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr; Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 97 is Ile, Val, Lys, Ala, or Asn; Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 101 is Asp; Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 103 is Asp, or Ser; Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro; Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp; Xaa at position 111 is Leu, Ile, Arg, Asp, or Met; Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 116 is Lys; Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus;(d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.
6. A method for the treatment of a patient having a hematopoietic disorder, comprising the steps of;(a) removing stem cells from said patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:19; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); whereinXaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly; Xaa at position 9 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at poSition 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 22 is Asp, Leu, or Val; Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 24 is Asn, or Ala; Xaa at position 26 is Leu, Trp, or Arg; Xaa at position 27 is Asn, Cys, Arg, Leu, His, Met, Pro; Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, Phe, Tyr, Ile or Met; Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 30 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, Ser, Ala, Ile, Glu, His or Trp; Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 33 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 34 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 43 is Asn or Gly; Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 45 is Glu Tyr; His, Leu, Pro, or Arg; Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 49 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 49 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp; Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 71 is Leu, Asn, Val, or Gln; Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 73 is Leu, Ser, Trp, or Gly; Xaa at position 74 is Ala, Lys, Arg, Val, or Trp; Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 79 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala or Pro; Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile or Tyr; Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 83 is Ile, Val, Lys, Ala, or Asn; Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 87 is Asp; Xaa at position 88 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 89 is Asp, or Ser; Xaa at position 90 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro; Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala or Trp; Xaa at position 97 is Leu, Ile, Arg, Asp, or Met; Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 102 is Lys; Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding native amino acids of (1-133) human interleukin-3;(d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.
7. A method for the treatment of a patient having a hematopoietic disorder, comprising the steps of;(a) removing stem cells from the patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:129; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 18 is Asn or Ile; Xaa at position 19 is Met, Ala or Ile; Xaa at position 20 is Ile, Pro or Leu; Xaa at position 23 is Ile, Ala or Leu; Xaa at position 25 is Thr or His; Xaa at position 29 is Gln, Arg, Val or Ile; Xaa at position 32 is Leu, Ala, Asn or Arg; Xaa at position 34 is Leu or Ser; Xaa at position 37 is Phe, Pro, or Ser; Xaa at position 38 is Asn or Ala; Xaa at position 42 is Gly, Ala, Ser, Asp or Asn; Xaa at position 45 is Gln, Val, or Met; Xaa at position 46 is Asp or Ser; Xaa at position 49 is Met, Ile, Leu or Asp; Xaa at position 50 is Glu or Asp; Xaa at position 51 is Asn Arg or Ser; Xaa at position 55 is Arg, Leu, or Thr; Xaa at position 56 is Pro or Ser; Xaa at position 59 is Glu or Leu; Xaa at position 60 is Ala or Ser; Xaa at position 62 is Asn, Val or Pro; Xaa at position 63 is Arg or His; Xaa at position 65 is Val or Ser; Xaa at position 67 is Ser, Asn, His or Gln; Xaa at position 69 is Gln or Glu; Xaa at position 73 is Ala or Gly; Xaa at position 76 is Ser, Ala or Pro; Xaa at position 79 is Lys, Arg or Ser; Xaa at position 82 is Leu, Glu, Val or Trp; Xaa at position 85 is Leu or Val; Xaa at position 87 is Leu, Ser, Tyr; Xaa at position 88 is Ala or Trp; xaa at position 91 is Ala or Pro; Xaa at position 93 is Pro or Ser; Xaa at position 95 is His or Thr; Xaa at position 98 is His, Ile, or Thr; Xaa at position 100 is Lys or Arg; Xaa at position 101 is Asp, Ala; Xaa at position 105 is Asn or Glu; Xaa at position 109 is Arg, Glu or Leu; Xaa at position 112 is Thr or Gln; Xaa at position 116 is Lys, Val, Xaa at position 117 is Thr or Ser; Xaa at position 120 is Asn, Gln, or His; Xaa at position 123 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3 and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus;(d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.
8. A method for the treatment of a patient having a hematopoietic disorder, comprising the steps of;(a) removing stem cells from said patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:30; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 4 is Asn or Ile; Xaa at position 5 is Met, Ala or Ile: Xaa at position 6 is Ile, Pro or Leu; Xaa at position 9 is Ile, Ala or Leu; Xaa at position 11 is Thr or His; Xaa at position 15 is Gln, Arg, Val or Ile; Xaa at position 18 is Leu, Ala, Asn or Arg; Xaa at position 20 is Leu or Ser; Xaa at position 23 is Phe, Pro, or Ser; Xaa at position 24 is Asn or Ala; Xaa at position 28 is Gly, Ala, Ser, Asp or Asn; Xaa at position 31 is Gln, Val, or Met; Xaa at position 32 is Asp or Ser; Xaa at position 35 is Met, Ile or Asp; Xaa at position 36 is Glu or Asp; Xaa at position 37 is Asn, Arg or Ser; Xaa at position 41 is Arg, Leu, or Thr; Xaa at position 42 is Pro or Ser; Xaa at position 45 is Glu or Leu; Xaa at position 46 is Ala or Ser; Xaa at position 48 is Asn, Val or Pro; Xaa at position 49 is Arg or His; Xaa at position 51 is Val or Ser; Xaa at position 53 is Ser, Asn, His or Gln; Xaa at position 55 is Gln or Glu; Xaa at position 59 is Ala or Gly; Xaa at position 62 is Ser, Ala or Pro; Xaa at position 65 is Lys, Arg or Ser; Xaa at position 67 is Leu, Glu, or Val; Xaa at position 68 is Leu, Glu, Val or Trp; Xaa at position 71 is Leu or Val; Xaa at position 73 is Leu, Ser or Tyr; Xaa at position 74 is Ala or Trp; Xaa at position 77 is Ala or Pro; Xaa at position 79 is Pro or Ser; Xaa at position 81 is His or Thr; Xaa at position 84 is His, Ile, or Thr; Xaa at position 86 is Lys or Arg; Xaa at position 87 is Asp, Ala; Xaa at position 91 is Asn or Glu; Xaa at position 95 is Arg, Glu Leu; Xaa at position 98 Thr or Gln; Xaa at position 102 is Lys, Val; Xaa at position 103 is Thr or Ser; Xaa at position 106 is Asn, Gln, or His; Xaa at position 109 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3;(d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.
9. The method according to claim 1, 2, 3, 4, 5, 6, 7, or 8, wherein said modified human interleukin-3 polypeptide has at least five times greater cell proliferative activity than native human interleukin-3.
10. The method according to claim 1, 2, 3, 4, 5, 6, 7, or 8, wherein said modified human interleukin-3 polypeptide has at least ten times greater cell proliferative activity than native human interleukin-3.
11. The method according to claim 2 or 6, wherein said mutant human interleukin-3 polypeptide is selected from the group consisting of:(a) a polypeptide having an amino acid sequence of (i) residues 3-113 of SEQ ID NO:66; (ii) residues 3-113 of SEQ ID NO:67; (iii) residues 3-113 of SEQ ID NO:69; or (iv) SEQ ID NO:218; and (b) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (a).
12. A method for selective ex-vivo expansion of stem cells, consisting of the steps of;(a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:15; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); whereinXaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly; Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 36 is Asp, Leu, or Val; Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 38 is Asn, or Ala; Xaa at position 40 is Leu, Trp, or Arg; Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro; Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala; Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His; Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 57 is Asn or Gly; Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg; Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp; Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 85 is Leu, Asn, Val, or Gln; Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 87 is Leu, Ser, Trp, or Gly; Xaa at position 88 is Ala, Lys, Arg, Val, or Trp; Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro; Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr; Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 97 is Ile, Val, Lys, Ala, or Asn; Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 101 is Asp; Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 103 is Asp, or Ser; Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro; Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp; Xaa at position 111 is Leu, Ile, Arg, Asp, or Met; Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 116 is Lys; Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus; and(c) harvesting said cultured cells.
13. A method for selective ex-vivo expansion of stem cells, consisting of the steps of;(a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:19; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); whereinXaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly; Xaa at position 9 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 22 is Asp, Leu, or Val; Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 24 is Asn, or Ala; Xaa at position 26 is Leu, Trp, or Arg; Xaa at position 27 is Asn, Cys, Arg, Leu, His, Met, Pro; Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, Phe, Tyr, Ile or Met; Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 30 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, Ser, Ala, Ile, Glu, His or Trp; Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at PoSition 33 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 34 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 43 is Asn or Gly; Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 45 is Glu Tyr, His, Leu, Pro, or Arg; Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 48 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 49 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp; Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 71 is Leu, Asn, Val, or Gln; Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 73 is Leu, Ser, Trp, or Gly; Xaa at position 74 is Ala, Lys, Arg, Val, or Trp; Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 79 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala or Pro; Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile or Tyr; Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 83 is Ile, Val, Lys, Ala, or Asn; Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 87 is Asp; Xaa at position 88 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 89 is Asp, or Ser; Xaa at position 90 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro; Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala or Trp; Xaa at position 97 is Leu, Ile, Arg, Asp, or Met; Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 102 is Lys; Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding native amino acids of (1-133) human interleukin-3; and(c) harvesting said cultured cells.
14. A method for selective ex-vivo expansion of stem cells, consisting of the steps of;(a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:129; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 18 is Asn or Ile; Xaa at position 19 is Met, Ala or Ile; Xaa at position 20 is Ile, Pro or Leu; Xaa at position 23 is Ile, Ala or Leu; Xaa at position 25 is Thr or His; Xaa at position 29 is Gln, Arg, Val or Ile; Xaa at position 32 is Leu, Ala, Asn or Arg; Xaa at position 34 is Leu or Ser; Xaa at position 37 is Phe, Pro, or Ser; Xaa at position 38 is Asn or Ala; Xaa at position 42 is Gly, Ala, Ser, Asp or Asn; Xaa at position 45 is Gln, Val, or Met; Xaa at position 46 is Asp or Ser; Xaa at position 49 is Met, Ile, Leu or Asp; Xaa at position 50 is Glu or Asp; Xaa at position 51 is Asn Arg or Ser; Xaa at position 55 is Arg, Leu, or Thr; Xaa at position 56 is Pro or Ser; Xaa at position 59 is Glu or Leu; Xaa at position 60 is Ala or Ser; Xaa at position 62 is Asn, Val or Pro; Xaa at position 63 is Arg or His; Xaa at position 65 is Val or Ser; Xaa at position 67 is Ser, Asn, His or Gln; Xaa at position 69 is Gln or Glu; Xaa at position 73 is Ala or Gly; Xaa at position 76 is Ser, Ala or Pro; Xaa at position 79 is Lys, Arg or Ser; Xaa at position 82 is Leu, Glu, Val or Trp; Xaa at position 85 is Leu or Val; Xaa at position 87 is Leu, Ser, Tyr; Xaa at position 88 is Ala or Trp; Xaa at position 91 is Ala or Pro; Xaa at position 93 is Pro or Ser; Xaa at position 95 is His or Thr; Xaa at position 98 is His, Ile, or Thr; Xaa at position 100 is Lys or Arg; Xaa at position 101 is Asp, Ala; Xaa at position 105 is Asn or Glu; Xaa at position 109 is Arg, Glu or Leu; Xaa at position 112 is Thr or Gln; Xaa at position 116 is Lys, Val, Xaa at position 117 is Thr or Ser; Xaa at position 120 is Asn, Gln, or His; Xaa at position 123 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3; and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus; and(c) harvesting said cultured cells.
15. A method for selective ex-vivo expansion of stem cells, consisting of the steps of;(a) separating stem cells from other cells; (b) culturing the separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:130; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 4 is Asn or Ile; Xaa at position 5 is Met, Ala or Ile: Xaa at position 6 is Ile, Pro or Leu; Xaa at position 9 is Ile, Ala or Leu; Xaa at position 11 is Thr or His; Xaa at position 15 is Gln, Arg, Val or Ile; Xaa at position 18 is Leu, Ala, Asn or Arg; Xaa at position 20 is Leu or Ser; Xaa at position 23 is Phe, Pro, or Ser; Xaa at position 24 is Asn or Ala; Xaa at position 28 is Gly, Ala, Ser, Asp or Asn; Xaa at position 31 is Gln, Val, or Met; Xaa at position 32 is Asp or Ser; Xaa at position 35 is Met, Ile or Asp; Xaa at position 36 is Glu or Asp; Xaa at position 37 is Asn, Arg or Ser; Xaa at position 41 is Arg, Leu, or Thr; Xaa at position 42 is Pro or Ser; Xaa at position 45 is Glu or Leu; Xaa at position 46 is Ala or Ser; Xaa at position 48 is Asn, Val or Pro; Xaa at position 49 is Arg or His; Xaa at position 51 is Val or Ser; Xaa at position 53 is Ser, Asn, His or Gln; Xaa at position 55 is Gln or Glu; Xaa at position 59 is Ala or Gly; Xaa at position 62 is Ser, Ala or Pro; Xaa at position 65 is Lys, Arg or Ser; Xaa at position 67 is Leu, Glu, or Val; Xaa at position 68 is Leu, Glu, Val or Trp; Xaa at position 71 is Leu or Val; Xaa at position 73 is Leu, Ser or Tyr; Xaa at position 74 is Ala or Trp; Xaa at position 77 is Ala or Pro; Xaa at position 79 is Pro or Ser; Xaa at position 81 is His or Thr; Xaa at position 84 is His, Ile, or Thr; Xaa at position 86 is Lys or Arg; Xaa at position 87 is Asp, Ala; Xaa at position 91 is Asn or Glu; Xaa at position 95 is Arg, Glu Leu; Xaa at position 98 Thr or Gln; Xaa at position 102 is Lys, Val; Xaa at position 103 is Thr or Ser; Xaa at position 106 is Asn, Gln, or His; Xaa at position 109 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3; and(c) harvesting said cultured cells.
16. A method for the treatment of a patient having a hematopoietic disorder, consisting of the steps of;(a) removing stem cells from said patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:15; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); whereinXaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly; Xaa at position 23 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 36 is Asp, Leu, or Val; Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 38 is Asn, or Ala; Xaa at position 40 is Leu, Trp, or Arg; Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro; Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala; Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His; Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 57 is Asn or Gly; Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg; Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp; Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 85 is Leu, Asn, Val, or Gln; Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 87 is Leu, Ser, Trp, or Gly; Xaa at position 88 is Ala, Lys, Arg, Val, or Trp; Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro; Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr; Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 97 is Ile, Val, Lys, Ala, or Asn; Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 101 is Asp; Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; Xaa at position 103 is Asp, or Ser; Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro; Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp; Xaa at position 111 is Leu, Ile, Arg, Asp, or Met; Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 116 is Lys; Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus; and(e) transplanting said cultured cells into said patient.
17. A method for the treatment of a patient having a hematopoietic disorder, consisting of the steps of;(a) removing stem cells from said patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:19; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); whereinXaa at position 3 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; Xaa at position 4 is Asn, His, Leu, Ile, Phe, Arg, or Gln; Xaa at position 5 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; Xaa at position 6 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; Xaa at position 7 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; Xaa at position 8 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val, or Gly; Xaa at position 9 is Ile, Val, Ala, Leu, Gly, Trp, Lys, Phe, Ser, or Arg; Xaa at position 10 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; Xaa at position 11 is Thr, His, Gly, Gln, Arg, Pro, or Ala; Xaa at position 12 is His, Thr, Phe, Gly, Arg, Ala, or Trp; Xaa at position 13 is Leu, Gly, Arg, Thr, Ser, or Ala; Xaa at position 14 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; Xaa at position 15 is Gln, Asn, Leu, Pro, Arg, or Val; Xaa at position 16 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; Xaa at position 17 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; Xaa at position 18 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; Xaa at position 19 is Pro, Leu, Gln, Ala, Thr, or Glu; Xaa at position 20 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; Xaa at position 21 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; Xaa at position 22 is Asp, Leu, or Val; Xaa at position 23 is Phe, Ser, Pro, Trp, or Ile; Xaa at position 24 is Asn, or Ala; Xaa at position 26 is Leu, Trp, or Arg; Xaa at position 27 is Asn, Cys, Arg, Leu, His, Met, Pro; Xaa at position 28 is Gly, Asp, Ser, Cys, Ala, Lys, Asn, Thr, Leu, Val, Glu, Phe, Tyr, Ile or Met; Xaa at position 29 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; Xaa at position 30 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; Xaa at position 31 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Asp, Asn, Arg, Ser, Ala, Ile, Glu, His or Trp; Xaa at position 32 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; Xaa at position 33 is Ile, Gly, Val, Ser, Arg, Pro, or His; Xaa at position 34 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; Xaa at position 35 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; Xaa at position 36 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; Xaa at position 37 is Asn, Arg, Met, Pro, Ser, Thr, or His; Xaa at position 38 is Asn, His, Arg, Leu, Gly, Ser, or Thr; Xaa at position 39 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; Xaa at position 40 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; Xaa at position 41 is Arg, Thr, Val, Ser, Leu, or Gly; Xaa at position 42 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; Xaa at position 43 is Asn or Gly; Xaa at position 44 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; Xaa at position 45 is Glu, Tyr, His, Leu, Pro, or Arg; Xaa at position 46 is Ala, Ser, Pro, Tyr, Asn, or Thr; Xaa at position 47 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; Xaa at position 48 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; Xaa at position 49 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; Xaa at position 50 is Ala, Asn, Pro, Ser, or Lys; Xaa at position 51 is Val, Thr, Pro, His, Leu, Phe, or Ser; Xaa at position 52 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; Xaa at position 53 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; Xaa at position 54 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; Xaa at position 55 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; Xaa at position 56 is Asn, Leu, Val, Trp, Pro, or Ala; Xaa at position 57 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; Xaa at position 58 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; Xaa at position 59 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; Xaa at position 60 is Ile, Met, Thr, Pro, Arg, Gly, Ala; Xaa at position 61 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; Xaa at position 62 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; Xaa at position 63 is Ile, Ser, Arg, Thr, or Leu; Xaa at position 64 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; Xaa at position 65 is Lys, Thr, Gly, Asn, Met, Arg, Ile, or Asp; Xaa at position 66 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; Xaa at position 67 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; Xaa at position 68 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; Xaa at position 69 is Pro, Ala, Thr, Trp, Arg, or Met; Xaa at position 70 is Cys, Glu, Gly, Arg, Met, or Val; Xaa at position 71 is Leu, Asn, Val, or Gln; Xaa at position 72 is Pro, Cys, Arg, Ala, or Lys; Xaa at position 73 is Leu, Ser, Trp, or Gly; Xaa at position 74 is Ala, Lys, Arg, Val, or Trp; Xaa at position 75 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; Xaa at position 76 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; Xaa at position 77 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; Xaa at position 78 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; Xaa at position 79 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; Xaa at position 80 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala or Pro; Xaa at position 81 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile or Tyr; Xaa at position 82 is Pro, Lys, Tyr, Gly, Ile, or Thr; Xaa at position 83 is Ile, Val, Lys, Ala, or Asn; Xaa at position 84 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; Xaa at position 85 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; Xaa at position 86 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; Xaa at position 87 is Asp; Xaa at position 89 is Asp, or Ser; Xaa at position 90 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; Xaa at position 91 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; Xaa at position 92 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; Xaa at position 94 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala, or Pro; Xaa at position 95 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; Xaa at position 96 is Lys, Asn, Thr, Leu, Gln, Arg, His, Glu, Ser, Ala or Trp; Xaa at position 97 is Leu, Ile, Arg, Asp, or Met; Xaa at position 98 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; Xaa at position 99 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; Xaa at position 100 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; Xaa at position 101 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; Xaa at position 102 is Lys; Xaa at position 103 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; Xaa at position 104 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; Xaa at position 105 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; Xaa at position 106 is Asn, Ala, Pro, Leu, His, Val, or Gln; Xaa at position 107 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; Xaa at position 108 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; Xaa at position 109 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; wherein from 1 to 3 of the amino acids designated by Xaa are different from the corresponding native amino acids of (1-133) human interleukin-3;(d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.
18. A method for the treatment of a patient having a hematopoietic disorder, consisting of the steps of;(a) removing stem cells from the patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:129; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 18 is Asn or Ile; Xaa at position 19 is Met, Ala or Ile; Xaa at position 20 is Ile, Pro or Leu; Xaa at position 23 is Ile, Ala or Leu; Xaa at position 25 is Thr or His; Xaa at position 29 is Gln, Arg, Val or Ile; Xaa at position 32 is Leu, Ala, Asn or Arg; Xaa at position 34 is Leu or Ser; Xaa at position 37 is Phe, Pro, or Ser; Xaa at position 38 is Asn or Ala; Xaa at position 42 is Gly, Ala, Ser, Asp or Asn; Xaa at position 45 is Gln, Val, or Met; Xaa at position 46 is Asp or Ser; Xaa at position 49 is Met, Ile, Leu or Asp; Xaa at position 50 is Glu or Asp; Xaa at position 51 is Asn Arg or Ser; Xaa at position 55 is Arg, Leu, or Thr; Xaa at position 56 is Pro or Ser; Xaa at position 59 is Glu or Leu; Xaa at position 60 is Ala or Ser; Xaa at position 62 is Asn, Val or Pro; Xaa at position 63 is Arg or His; Xaa at position 65 is Val or Ser; Xaa at position 67 is Ser, Asn, His or Gln; Xaa at position 69 is Gln or Glu; Xaa at position 73 is Ala or Gly; Xaa at position 76 is Ser, Ala or Pro; Xaa at position 79 is Lys, Arg or Ser; Xaa at position 82 is Leu, Glu, Val or Trp; Xaa at position 85 is Leu or Val; Xaa at position 87 is Leu, Ser, Tyr; Xaa at position 88 is Ala or Trp; Xaa at position 91 is Ala or Pro; Xaa at position 93 is Pro or Ser; Xaa at position 95 is His or Thr; Xaa at position 98 is His, Ile, or Thr; Xaa at position 100 is Lys or Arg; Xaa at position 101 is Asp, Ala; Xaa at position 105 is Asn or Glu; Xaa at position 109 is Arg, Glu or Leu; Xaa at position 112 is Thr or Gln; Xaa at position 116 is Lys, Val; Xaa at position 117 is Thr or Ser; Xaa at position 120 is Asn, Gln, or His; Xaa at position 123 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3 and wherein from 1 to 14 amino acids are optionally deleted from the N-terminus and/or from 1 to 15 amino acids are optionally deleted from the C-terminus;(d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.
19. A method for the treatment of a patient having a hematopoietic disorder, consisting of the steps of;(a) removing stem cells from said patient; (b) separating said stem cells from other cells; (c) culturing said separated stem cells in a growth media comprising; a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and Methylcellulose assay said polypeptide comprising a sequence; selected from the group consisting of: (i) the sequence of SEQ ID NO:130; and (ii) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (i); wherein Xaa at position 4 is Asn or Ile; Xaa at position 5 is Met, Ala or Ile: Xaa at position 6 is Ile, Pro or Leu; Xaa at position 9 is Ile, Ala or Leu; Xaa at position 11 is Thr or His; Xaa at position 15 is Gln, Arg, Val or Ile; Xaa at position 18 is Leu, Ala, Asn or Arg; Xaa at position 20 is Leu or Ser; Xaa at position 23 is Phe, Pro, or Ser; Xaa at position 24 is Asn or Ala; Xaa at position 28 is Gly, Ala, Ser, Asp or Asn; Xaa at position 31 is Gln, Val, or Met; Xaa at position 32 is Asp or Ser; Xaa at position 35 is Met, Ile or Asp; Xaa at position 36 is Glu or Asp; Xaa at position 37 is Asn, Arg or Ser; Xaa at position 41 is Arg, Leu, or Thr; Xaa at position 42 is Pro or Ser; Xaa at position 45 is Glu or Leu; Xaa at position 46 is Ala or Ser; Xaa at position 48 is Asn, Val or Pro; Xaa at position 49 is Arg or His; Xaa at position 51 is Val or Ser; Xaa at position 53 is Ser, Asn, His or Gln; Xaa at position 55 is Gln or Glu; Xaa at position 59 is Ala or Gly; Xaa at position 62 is Ser, Ala or Pro; Xaa at position 65 is Lys, Arg or Ser; Xaa at position 67 is Leu, Glu, or Val; Xaa at position 68 is Leu, Glu, Val or Trp; Xaa at position 71 is Leu or Val; Xaa at position 73 is Leu, Ser or Tyr; Xaa at position 74 is Ala or Trp; Xaa at position 77 is Ala or Pro; Xaa at position 79 is Pro or Ser; Xaa at position 81 is His or Thr; Xaa at position 84 is His, Ile, or Thr; Xaa at position 86 is Lys or Arg; Xaa at position 87 is Asp, Ala; Xaa at position 91 is Asn or Glu; Xaa at position 95 is Arg, Glu Leu; Xaa at position 98 Thr or Gln; Xaa at position 102 is Lys, Val, Trp or Ser; Xaa at position 103 is Thr or Ser; Xaa at position 106 is Asn, Gln, or His; Xaa at position 109 is Ala or Glu; with the proviso that from one to three of the amino acids designated by Xaa are different from the corresponding amino acids of native human interleukin-3;(d) harvesting said cultured cells; and (e) transplanting said cultured cells into said patient.
20. The method according to claim 12, 13, 14, 15, 16, 17, 18, or 19, wherein said modified human interleukin-3 polypeptide has at least five times greater cell proliferative activity than native human interleukin-3.
21. The method according to claim 12, 13, 14, 15, 16, 17, 18, or 19 wherein said modified human interleukin-3 polypeptide has at least ten times greater cell proliferative activity than native human interleukin-3.
22. The method according to claim 13 or 17, wherein said mutant human interleukin-3 polypeptide is selected from the group consisting of:(a) a polypeptide having an amino acid sequence of (i) residues 3-113 of SEQ ID NO:66; (ii) residues 3-113 of SEQ ID NO:67; (iii) residues 3-113 of SEQ ID NO:69; or (iv) SEQ ID NO:218; and (b) an N-terminal methionine residue, alanine residue or methionine-alanine di-peptide immediately preceding said sequence according to (a).

Parent Case Info

This application is a continuation of U.S. application Ser. No. 08/411,796, filed Apr. 6, 1995, now U.S. Pat. No. 5,677,149; which entered the U.S. national stage under 35 USC §371 from PCT/US93/11198, filed Nov. 22, 1993; which is a continuation-in-part of U.S. application Ser. No. 07/981,044, filed Nov. 24, 1992, now abandoned. This is a continuation-in-part of U.S. application Ser. No. 07/981,044 filed Nov. 24, 1992 which is incorporated herein by reference.

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Continuations (1)

	Number	Date	Country
Parent	08/411796		US
Child	08/559390		US

Continuation in Parts (1)

	Number	Date	Country
Parent	07/981044	Nov 1992	US
Child	08/411796		US

Methods of using interleukin-3 (IL-3) mutant polypeptides for ex-vivo expansion of hematopoietic stem cells

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Abstract

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US Referenced Citations (6)

Foreign Referenced Citations (15)

Non-Patent Literature Citations (17)

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Continuation in Parts (1)