Nematode-extracted anticoagulant protein

FIELD OF THE INVENTION
The present invention relates to specific proteins as well as recombinant versions of these proteins which are potent anticoagulants in human plasma. These proteins include certain proteins extracted from nematodes. In another aspect, the present invention relates to compositions comprising these proteins, which are useful as potent and specific inhibitors of blood coagulation in vitro and in vivo, and methods for their use as in vitro diagnostic agents, or as in vivo therapeutic agents, to prevent the clotting of blood. In a further aspect, the invention relates to nucleic acid sequences, including mRNA and DNA, encoding the proteins and their use in vectors to transfect or transform host cells and as probes to isolate certain related genes in other species and organisms.
BACKGROUND AND INTRODUCTION TO THE INVENTION
Normal hemostasis is the result of a delicate balance between the processes of clot formation (blood coagulation) and clot dissolution (fibrinolysis). The complex interactions between blood cells, specific plasma proteins and the vascular surface, maintain the fluidity of blood unless injury occurs. Damage to the endothelial barrier lining the vascular wall exposes underlying tissue to these blood components. This in turn triggers a series of biochemical reactions altering the hemostatic balance in favor of blood coagulation which can either result in the desired formation of a hemostatic plug stemming the loss of blood or the undesirable formation of an occlusive intravascular thrombus resulting in reduced or complete lack of blood flow to the affected organ.
The blood coagulation response is the culmination of a series of amplified reactions in which several specific zymogens of serine proteases in plasma are activated by limited proteolysis. This series of reactions results in the formation of an insoluble matrix composed of fibrin and cellular components which is required for the stabilization of the primary hemostatic plug or thrombus. The initiation and propagation of the proteolytic activation reactions occurs through a series of amplified pathways which are localized to membranous surfaces at the site of vascular injury (Mann, K. G., Nesheim, M. E., Church, W. R., Haley, P. and Krishnaswamy, S. (1990) Blood 76: 1-16, and Lawson, J. H., Kalafatis, M., Stram, S., and Mann, K. G. (1994) J. Biol. Chem. 269: 23357-23366).
Initiation of the blood coagulation response to vascular injury follows the formation of a catalytic complex composed of serine protease factor VIIa and the non-enzymatic co-factor, tissue factor (TF)(Rappaport, S. I. and Rao, L. V. M. (1992) Arteriosclerosis and Thrombosis 12: 1112-1121). This response appears to be exclusively regulated by the exposure of subendothelial TF to trace circulating levels of factor VIIa and its zymogen factor VII, following a focal breakdown in vascular integrity. Autoactivation results in an increase in the number of factor VIIa/TF complexes which are responsible for the formation of the serine protease factor Xa. It is believed that in addition to the factor VIIa/TF complex, the small amount of factor Xa which is formed primes the coagulation response through the proteolytic modification of factor IX to factor IX.sub.alpha which in turn is converted to the active serine protease factor IXa.sub.b by the factor VIIa/TF complex (Mann, K. G., Krishnaswamy, S. and Lawson, J. H. (1992) Sem. Hematology 29: 213-226.). It is factor IXa.sub.b in complex with activated factor VIIIa, which appears to be responsible for the production of significant quantities of factor Xa which subsequently catalyzes the penultimate step in the blood coagulation cascade; the formation of the serine protease thrombin.
Factor Xa catalyzes the formation of thrombin following the assembly of the prothrombinase complex which is composed of factor Xa, the non-enzymatic co-factor Va and the substrate prothrombin (factor II) assembled in most cases, on the surface of activated platelets which are adhered at the site of injury (Fuster, V., Badimon, L., Badimon, J. J. and Chesebro, J. H. (1992) New Engl. J. Med. 326: 310-318). In the arterial vasculature, the resulting amplified "burst" of thrombin generation catalyzed by prothrombinase results locally high levels of this protease which is responsible for the formation of fibrin and the further recruitment of additional platelets as well as the covalent stabilization of the clot through the activation of the transglutaminase zymogen factor XIII. In addition, the coagulation response is further propagated through the throibin-mediated proteolytic feedback activation of the non-enzymatic co-factors V and VIII resulting in more prothrombinase formation and subsequent thrombin generation (Hemker, H. C. and Kessels, H. (1991) Haemostasis 21: 189-196).
Substances which interfere in the process of blood coagulation (anticoagulants) have been demonstrated to be important therapeutic agents in the treatment and prevention of thrombotic disorders (Kessler, C. M. (1991) Chest 99: 97S-112S and Cairns, J. A., Hirsh, J., Lewis, H. D., Resnekov, L., and Theroux, P. (1992) Chest 102: 456S481S). The currently approved clinical anticoagulants have been associated with a number of adverse effects owing to the relatively non-specific nature of their effect on the blood coagulation cascade (Levine, M. N., Hirsh, J., Landefeld, S., and Raskob, G. (1992) Chest 102: 352S-363S). This has stimulated the search for more effective anticoagulant agents which can more effectively control the activity of the coagulation cascade by selectively interfering with specific reactions in this process which may have a positive effect in reducing the complications of anticoagulant therapy (Weitz, J., and Hirsh, J. (1993) J. Lab. Clin. Med. 122: 364-373). In another aspect, this search has focused on normal human proteins which serve as endogenous anticoagulants in controlling the activity of the blood coagulation cascade. In addition, various hematophageous organisms have been investigated because of their ability to effectively anticoagulate the blood meal during and following feeding on their hosts suggesting that they have evolved effective anticoagulant strategies which may be useful as therapeutic agents.
A plasma protein, Lipoprotein-Associated Coagulation Inhibitor (LACI) or recently termed Tissue Factor Pathway Inhibitor (TFPI), containing three consecutive Kunitz domains has been reported to inhibit the enzyme activity of factor Xa directly and, in a factor Xa-dependent manner, inhibit the enzyme activity the factor VIIa-tissue factor complex. Salvensen, G., and Pizzo, S.V., "Proteinase Inhibitors: .alpha.-Macroglobulines, Serpins, and Kunis", Hemostasis and Thrombosis, Third Edition, pp. 251-253, J. B. Lippincott Company (Edit. R. W. Colman et al. 1994). A cDNA sequence encoding TFPI has been reported, and the cloned protein was reported to have a molecular weight of 31,950 daltons and contain 276 amino acids. Broze, G. J. and Girad, T. J., U.S. Pat. No. 5,106,833, col. 1, (1992). Various recombinant proteins derived from TFPI have been reported. Girad, T. J. and Broze, G. J., EP 439,442 (1991); Rasmussen, J. S. and Nordfand, O. J., WO 91/02753 (1991); and Broze, G. J. and Girad, T. J., U.S. Pat. No. 5,106,833, col. 1, (1992).
Antistasin, a protein comprised of 119 amino acids and found in the salivary gland of the Mexican leech, Haementeria officinalis, has been reported to inhibit the enzyme activity of factor Xa. Tuszynski et al., J. Biol. Chem, 262:9718 (1987); Nutt, et al., J. Biol. Chem, 263:10162 (1988). A 6,000 daltons recombinant protein containing 58 amino acids with a high degree homology to antistasin's amino-terminus amino acids 1 through 58 has been reported to inhibit the enzyme activity of factor Xa. Tung, J. et al., EP 454,372 (Oct. 30, 1991); Tung, J. et al., U.S. Pat. No. 5,189,019 (Feb. 23, 1993).
Tick Anticoagulant Protein (TAP), a protein comprised of 60 amino acids and isolated from the soft tick, Ornithodoros moubata, has been reported to inhibit the enzyme activity of factor Xa but not factor VIIA. Waxman, L. et al., Science, 248:593 (1990). TAP made by recombinant methods has been reported. Vlausk, G. P. et al., EP 419,099 (1991) and Vlausk, G. P. et al., U.S. Pat. No 5,239,058 (1993).
The dog hookworm, Ancylostoma caninum, which can also infect humans, has been reported to contain a potent anticoagulant substance. A. caninum was reported to contain substance which inhibited coagulation of blood in vitro. Loeb, L. and Smith, A. J., Proc. Pathol. Soc. Philadelphia, 7:173-178 (1904). Extracts of A. caninum were reported to prolong prothrombin time and partial thromboplastin time in human plasma with the anticoagulant effect being reported attributable to inhibition of factor Xa but not thrombin. Spellman, Jr., J. J. and Nossel H. L., Am. J. Physiol., 220:922-927 (1971). More recently, soluble protein extracts of A. caninum were reported to prolong prothrombin time and partial thromboplastin time in human plasma in vitro. The anticoagulant effect was reported to be attributable to inhibition of human factor Xa but not thrombin. Cappello, M, et al., J. Infect. Diseases, 167:1474-1477 (1993).
The human hookworm, Ancylostoma ceylanicum, has also been reported to contain an anticoagulant. Extracts of A. ceylanicum have been reported to prolong prothrombin time and partial thromboplastin time in dog and human plasma in vitro. Carroll, S. M., et al., Thromb. Haemostas. (Stuttgart), 51:222-227 (1984).
Soluble extracts of the parasite, Ascaris suum, have been reported to contain an anticoagulant. These extracts were reported to prolong the clotting of whole blood, as well as clotting time in the kaolin-activated partial thromboplastin time test but not in the prothrombin time test. Crawford, G. P. M. et al., J. Parasitol., 68: 1044-1047 (1982). Chymotrypsin/elastase inhibitor-1 and its major isoforms, trypsin inhibitor-1 and chymotrypsin/elastase inhibitor-4, isolated from Ascaris suum, were reported to be serine protease inhibitors and share a common pattern of five-disulfide bridges. Bernard, V. D. and Peanasky, R. J., Arch. Biochem. Biophys., 303:367-376 (1993); Huang, K. et al., Structure, 2:679-689 (1994); and Grasberger, B. L. et al., Structure, 2:669-678 (1994). There was no indication that the reported serine protease inhibitors had anticoagulant activity.
SUMMARY OF THE INVENTION
The present invention is directed to isolated proteins having anticoagulant activity and including at least one NAP domain. We refer to these proteins as Nematode-extracted Anticoagulant Proteins or "NAPs". "NAP domain" refers to a sequence of the isolated protein or NAP believed to have anticoagulant activity. The anticoagulant activity of these proteins may be assessed by their activities in increasing clotting time of human plasma in the prothrombin time (PT) and activated partial thromboplastin time (aPTT) assays. It is believed that the NAP domain is responsible for the observed anticoagulent activity of these proteins. Certain of these proteins have at least one NAP domain which is an amino acid sequence having a molecular weight of about 5.0 to 8.0 kilodaltons and containing 10 cysteine amino acid residues.
In another aspect, the present invention is directed to a method of preparing and isolating a recombinant cDNA molecule encoding a recombinant protein exhibiting anticoagulant activity and having a NAP domain, and to a cDNA molecule made by this method. This method comprises the steps of: (a) isolating a cDNA library from a species of nematode; (b) ligating said cDNA library into a cloning vector; (c) introducing said cloning vector containing said cDNA library into a host cell; (d) contacting the cDNA molecules of said host cell with a solution containing a hybridization probe having a nucleic acid sequence comprising AAR GCi TAY CCi GAR TGY GGi GAR AAY GAR TGG �SEQ. ID. NO. 1!, wherein R is A or G, Y is T or C, and i is inosine; (e) detecting a recombinant cDNA molecule which hybridizes to said probe; and (f) isolating said recombinant cDNA molecule.
In another aspect, the present invention is directed to method of making a recombinant protein which has anticoagulant activity and which includes a NAP domain and to recombinant proteins made by this method. This method comprises the steps of: (a) isolating a cDNA library from a species of nematode; (b) ligating said cDNA library into a cloning vector; (c) introducing said cloning vector containing said cDNA library into a first host cell; (d) contacting the cDNA molecules of said first host cell with a solution containing a hybridization probe having a nucleic acid sequence comprising AAR GCi TAY CCi GAR TGY GGi GAR AAY GAR TGG �SEQ. ID. NO. 1!, wherein R is A or G, Y is T or C, and i is inosine; (e) detecting a recombinant cDNA molecule which hybridizes to said probe; (f) isolating said recombinant cDNA molecule; (g) ligating the nucleic acid sequence of said cDNA molecule which encodes said recombinant protein into an expression vector; (h) transforming a second host cell with said expression vector containing said nucleic acid sequence of said cDNA molecule which encodes said recombinant protein; (i) culturing the transformed second host cell; and (j) isolating said recombinant protein expressed by said second host cell. It is noted that when describing production of recombinant proteins in certain expression systems such as COS cells, the term "transfection" is conventionally used in place of (and sometimes interchangeably with) "transformation".
In another aspect, the present invention is directed to a method of making a recombinant cDNA encoding a recombinant protein having anticoagulant activity and having a NAP domain, comprising the steps of: (a) isolating a cDNA library from a nematode;(b) ligating said cDNA library into a cloning vector; (c) introducing said cloning vector containing said cDNA library into a host cell; (d) contacting the cDNA molecules of said host cells with a solution comprising first and second hybridization probes, wherein said first hybridization probe has the nucleic acid sequence comprising AAG GCA TAC CCG GAG TGT GGT GAG AAT GAA TGG CTC GAC GAC TGT GGA ACT CAG AAG CCA TGC GAG GCC AAG TGC AAT GAG GAA CCC CCT GAG GAG GAA GAT CCG ATA TGC CGC TCA CGT GGT TGT TTA TTA CCT CCT GCT TGC GTA TGC AAA GAC GGA TTC TAC AGA GAC ACG GTG ATC GGC GAC TGT GTT AGG GAA GAA GAA TGC GAC CAA CAT GAG ATT ATA CAT GTC TGA �SEQ. ID. NO. 2!, and said second hybridization probe has the nucleic acid sequence comprising AAG GCA TAC CCG GAG TGT GGT GAG AAT GAA TGG CTC GAC GTC TGT GGA ACT AAG AAG CCA TGC GAG GCC AAG TGC AGT GAG GAA GAG GAG GAA GAT CCG ATA TGC CGA TCA TTT TCT TGT CCG GGT CCC GCT GCT TGC GTA TGC GAA GAC GGA TTC TAC AGA GAC ACG GTG ATC GGC GAC TGT GTT AAG GAA GAA GAA TGC GAC CAA CAT GAG ATT ATA CAT GTC TGA �SEQ. ID. NO. 3!; (e) detecting a recombinant cDNA molecule which hybridizes to said mixture of said probes; and (f) isolating said recombinant cDNA molecule.
In yet another aspect, the present invention is directed to a method of making a recombinant cDNA encoding a protein having anticoagulant activity and which encodes a NAP domain, comprising the steps of: (a) isolating a cDNA library from a nematode; (b) ligating said cDNA library into a phagemid vector; (c) transforming host cells with said vector containing said cDNA library; (d) culturing said host cells; (e) infecting said host cells with a helper phage; (f) separating phage containing said cDNA library from said host cells; (g) combining a solution of said phage containing said cDNA library with a solution of biotinylated human factor Xa; (h) contacting a streptavidin-coated solid phase with said solution containing said phages containing said cDNA library, and said biotinylated human factor Xa; (i) isolating phages which bind to said streptavidin-coated solid phase, and (j) isolating the recombinant cDNA molecule from phages which bind to said streptavidin-coated solid phase.
In one preferred aspect, the present invention is directed to a recombinant cDNA having a nucleic acid sequence selected from the nucleic acid sequences depicted in FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E and FIG. 7F.
In an alternate preferred aspect, the present invention is directed to a recombinant cDNA having the nucleic acid sequence depicted in FIG. 9.
DEFINITIONS
The term "amino acid" refers to the natural L-amino acids. Natural L-amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val).
The term "amino acid residue" refers to radicals having the structure: (1) --NH--CH(R)C(.dbd.O)--, wherein R is the alpha-carbon side-chain group of an L-amino acid, except for L-proline; or (2) ##STR1## for L-proline.
The term "peptide" refers to a sequence of amino acids linked together through their alpha-amino and carboxylate groups by peptide bonds. Such sequences as shown herein are presented in the amino to carboxy direction, from left to right.
The term "protein" refers to a molecule comprised of one or more peptides.
The term "cDNA" refers to complementary DNA.
The term "nucleic acid" refers to polymers in which bases (e.g., purines or pyrimidines) are attached to a sugar phosphate backbone. Nucleic acids include DNA and RNA.
The term "nucleic acid sequence" refers to the sequence of nucleosides comprising a nucleic acid. Such sequences as shown herein are presented in the 5' to 3' direction, from left to right.
The term "recombinant DNA molecule" refers to a DNA molecule created by ligating together pieces of DNA that are not normally continguous.
The term "mRNA" refers to messenger ribonucleic acid.
The term "homology" refers to the degree of similarity of DNA or peptide sequences.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the nucleotide sequence of the NAP5 cDNA �SEQ. ID. NO. 32!. The numbering starts at the first nucleotide of the cDNA. Translation starts at the first ATG codon (position 14); a second in frame ATG is present at position 20.
FIG. 2 depicts the amino acid sequence of mature NAP isoform 5 �SEQ. ID. NO. 33!.
FIG. 3 depicts the nucleotide sequence of the NAP6 cDNA �SEQ. ID. NO. 34!. The numbering starts at the first nucleotide of the cDNA. Translation starts at the first ATG codon (position 14); a second in frame ATG is present at position 20.
FIG. 4 depicts the amino acid sequence of mature NAP isoform 6 �SEQ. ID. NO. 35!. Amino acids that differ from NAP isoform 5 are underlined. In addition to these amino acid substitutions, NAP isoform 6 contains a two amino acid deletion (Pro--Pro) when compared to NAP isoform 5.
FIG. 5 depicts the amino acid sequence of Pro-NAP isoform 5 �SEQ. ID. NO. 36!.
FIG. 6 depicts the amino acid sequence of Pro-NAP isoform 6 �SEQ. ID. NO. 37!. Amino acids that differ from Pro-NAP isoform 5 are underlined. In addition to these amino acid substitutions, Pro-NAP isoform 6 contains a two amino acid deletion (Pro-Pro) when compared to Pro-NAP isoform 5.
FIGS. 7A through 7F depict the nucleotide sequences of the cDNAs and deduced amino acid sequences of certain NAP proteins isolated from Ancylostoma ceylanicum, Ancylostoma duodenale, and Heligmosomoides polygryrus. FIG. 7A depicts sequences for the recombinant cDNA molecule, AceNAP4, isolated from Ancylostoma ceylanicum �SEQ. ID. NO. 38!. FIG. 7B depicts sequences for the recombinant cDNA molecule, AceNAP5, isolated from Ancylostoma ceylanicum �SEQ. ID. NO. 39!. FIG. 7C depicts sequences for the recombinant cDNA molecule, AceNAP7, isolated from Ancylostom ceylanicum �SEQ. ID. NO. 40!. FIG. 7D depicts sequences for the recombinanct cDNA molecule, AduNAP4, isolated from Ancylostoma duodenale �SEQ. ID. NO. 41!. FIG. 7E depicts sequences for the recombinant cDNA molecule, AduNAP7, isolated from Ancylostoma duodenale �SEQ. ID. NO. 42!. FIG. 7F depicts sequences for the recombinant cDNA molecule, HpoNAP5, isolated from Heligmosomoides polygyrus �SEQ. ID. NO. 43!. The EcoRI site, corresponding to the 5'-end of the recombinant cDNA molecule, is indicated in all cases (underlined). Numbering of each sequence starts at this EcoRI site. AceNAP4 and AduNAP7, each encode a protein which has two NAP domains; all other clones code for a protein having a single NAP domain. The AduNAP4 cDNA clone is not full-length, i.e., the recombinant cDNA molecule lacks the 5'-terminal part of the coding region based on comparison with other isoforms.
FIGS. 8A through 8C depict the nucleotide sequence of the vectors, pDONG61 (FIG. 8A) �SEQ. ID. NO. 44!, pDONG62 (FIG. 8B) �SEQ. ID. NO. 45!, and pDONG63 (FIG. 8C) �SEQ. ID. NO. 46!. The HindIII-BamHI fragment which is shown is located between the HindIII and BamHI sites of pUC119. The vectors allow the cloning of cDNAs, as SfiI-NotI fragments, in the three different reading frames downstream of the filamentous phage gene 6. All relevant restriction sites are indicated. The AAA Lys-triplet at position 373-375 is the last codon of gene 6. The gene 6 encoded protein is followed by a Gly-Gly-Gly-Ser-Gly-Gly �SEQ. ID. NO. 4! linker sequence.
FIG. 9 depicts the nucleotide sequence of the recombinant cDNA molecule, NAPc2 cDNA �SEQ. ID. NO. 47!. The EcoRI site, corresponding to the 5'-end of the cDNA, is indicated (underlined). Numbering starts at this EcoRI site. The deduced amino acid sequence is also shown; the translational reading frame was determined by the gene 6 fusion partner. The NAPc2 cDNA lacks the 5'-terminal part of the coding region; the homology with the NAP isoforms 5 and 6 predicts that the first seven amino acid residues belong to the secretion signal.
FIGS. 10A and 10B depict the comparative effects of NAP proteins on the prothrombin time (PT) measurement (FIG. 10A) and the activated partial thromboplastin time (aPTT) (FIG. 10B) of normal citrated human plasma. Solid circles, (.circle-solid.), represent Pro-NAP isoform 5; open triangles, (.gradient.), represent NAP isoform 5; and open circles, (.smallcircle.), represent native NAP.
FIG. 11 depicts the alignment of the amino acid sequences encoded by NAP cDNAs isolated from various nematodes. NAP5 �SEQ. ID. NO. 48!, NAP6 �SEQ. ID. NO. 49!, and NAPc2 �SEQ. ID. NO. 50! were isolated from Ancylostoma caninum. AceNAP5 �SEQ. ID. NO. 51!, AceNAP7 �SEQ. ID. NO. 52!, and AceNAP4 �SEQ. ID. NO.s 53 and 54! were isolated from Ancylostoma ceylanicum. AduNAP4 �SEQ. ID. NO. 55! and AduNAP7 �SEQ. ID. NO.s 56 and 57! were isolated from Ancylostoma duodenale. HpoNAP5 �SEQ. ID. NO. 58! was isolated from Heligmosomoides polygrus. The amino acid sequences shown in this figure are as given in FIGS. 1, 3, 7A through 7F, and 9. The sequences of mature NAP isoforms 5 and 6 (see FIGS. 2 and 4) are characterized by ten cysteine residues (numbered one through ten and shown in bold). The region comprising these ten cysteines defines a NAP-domain. In addition to NAP5 and NAP6, all other derived amino acid sequences shown have at least one such NAP-domain. All of these amino acid sequences contain at least one NAP domain. The AceNAP4 cDNA consists of two adjacent regions, named AceNAP4d1 �SEQ. ID. NO. 53! and AceNAP4d2 �SEQ. ID NO. 54!, which encode a first (d1) and second (d2) NAP domain; similarly, the AduNAP7 cDNA contains two adjacent regions, AduNAP7d1 �SEQ. ID. NO. 56! and AduNAP7d2 �SEQ. ID. NO. 57!, encoding a first (d1) and second (d2) NAP-domain. The alignment of the amino acid sequences of all NAP-domains is guided by the cysteines; dashes ( - - - ) were introduced at certain positions to maintain the cysteine alignment and indicate the absence of an amino acid at that position. The carboxy-terminal residue of a cDNA encoded protein is followed by the word "end".
FIGS. 12A and 12B depict a map of the P. pastoris pYAM7SP8 expression/secretion vector (FIG. 12A) and sequences included in the vector (FIG. 12B) �SEQ. ID. NO. 59!. As depicted in FIG. 12A, this plasmid contains the following elements inserted between the methanol-induced AOX1 promoter (dark arrow in the 5'AOX untranslated region) and the AOX1 transcription termination signal (3'T): a synthetic DNA fragment encoding the acid phosphatase secretion signal (S), a synthetic 19-amino acid pro sequence (P) ending with a Lys-Arg processing site for the KEX2 protease and a multicloning site. The HIS4 gene which serves as a selection marker in GS115 transformation was modified by site directed mutagenesis to eliminate the Stu1 recognition sequence (HIS4*). pBR322 sequences, including the B1a gene and origin (ori) for propagation in E. coli are represented by a single line. FIG. 12B depicts the following contiguous DNA sequences which are incorporated in pYAM7SP8: the acid phosphatase (PHO1) secretion signal sequence, pro sequence and multicloning site (MCS) sequence. The ATG start codon of the PHO1 secretion signal is underlined.

DETAILED DESCRIPTION OF THE INVENTION
This invention provides a family of proteins, collectively referred to as Nematode-extracted Anticoagulant Proteins (NAPs). These proteins are so designated because the first member originally isolated was extracted from a nematode, the canine hookworm, Ancyclostoma caninum. However, the designation NAP or NAP domain should not be considered to limit the proteins of the present invention by this or other natural source.
Individual NAP proteins are characterized by having at least one NAP domain and by having anticoagulant activity. Such anticoagulant activity may be assessed by increases in clotting time in both the PT and aPTT assays described herein. The NAP domain is an amino acid sequence. It is believed that the NAP domain is responsible for the observed anticoagulant activity. Certain representative NAP domains include the amino acid sequences depicted in FIG. 11, particularly the sequences between the cysteines designated as Cysteine 1 and Cysteine 10 in FIG. 11. The characteristics broadly defining this family of proteins, as well as the nucleic acid molecules, including mRNAs sequences and DNA sequences which encode such proteins, are provided. Methods of making these proteins, as well as methods of making nucleic acid molecules encoding such proteins, are also provided. The specific examples provided are exemplary only and other members of the NAP family of proteins, as well as nucleic acid sequences encoding them, can be obtained by following the procedures outlined in these examples and described in herein.
The proteins of the present invention include isolated NAPs which comprise proteins having anticoagulant activity and including at least one NAP domain.
With respect to "anticoagulant activity", the purified proteins of the present invention are active as anticoagulants, and as such, are characterized by inhibiting the clotting of blood which includes the clotting of plasma. In one aspect, the preferred isolated proteins of the present invention include those which increase the clotting time of human plasma as measured in both the prothrombin time (PT) and activated partial thromboplastin time (aPTT) assays.
In the PT assay, clotting is initiated by the addition of a fixed amount of tissue factor-phospholipid micelle complex (thromboplastin) to human plasma. Substances acting as anticoagulants interfere with certain interactions on the surface of this complex and increase the time required to achieve a certain amount of clotting relative to the clotting observed in the absence of the anticoagulant. In the APTT assay, clotting is initiated by the addition of a certain fixed amount of negatively charged phospholipid micelle (activator) to the human plasma. Substances acting as anticoagulants will interfere with certain interactions on the surface of the complex and again increase the time to achieve a certain amount of clotting relative to that observed in the absence of the anticoagulant. Example B describes an example of such PT and APTT assays. These assays can be used to assess anticoagulant activity of the isolated NAPs of the present invention.
The preferred isolated NAPs of the present invention include those which double the clotting time of human plasma in the PT assay when present at a concentration of about 1 to about 500 nanomolar and which also double the clotting time of human plasma in the aPTT assay when present at a concentration of about 1 to about 500 nanomolar. Especially preferably are those proteins which double the clotting time of human plasma in the PT assay when present at a concentration of about 5 to about 100 nanomolar, and which also double the clotting time of human plasma in the aPTT assay when present at a concentration of about 5 to about 200 nanomolar. More especially preferred are those proteins which double the clotting time of human plasma in the PT assay when present at a concentration about 10 to about 50 nanomolar, and which also double the clotting time of human plasma in the aPTT assay when present at a concentration of about 10 to about 100 nanomolar.
With respect to "NAP domain", the isolated proteins (or NAPs) of the present invention include at least one NAP domain in their amino acid sequence. Certain NAP domains have an amino acid sequence having a molecular weight of about 5.0 to 8.0 kilodaltons and containing 10 cysteine amino acid residues.
Certain preferred isolated NAPs of the present invention include those which contain at least one NAP domain, wherein each such NAP domain is further characterized by including the amino acid sequence: Cys-A.sub.1 -Cys-A.sub.2 -Cys-A.sub.3 -Cys-A.sub.4 -Cys-A.sub.5 -Cys-A.sub.6 -Cys-A.sub.7 -Cys-A.sub.8 -Cys-A.sub.9 -Cys, wherein: (a) A.sub.1 is an amino acid sequence containing 7 to 8 amino acid residues; (b) A.sub.2 is an amino acid sequence containing 2 to 5 amino acid residues; (c) A.sub.3 is an amino acid sequence containing 3 amino acid residues; (d) A.sub.4 is an amino acid sequence containing 6 to 17 amino acid residues; (e) A.sub.5 is an amino acid sequence containing 3 to 4 amino acid residues; (f) A.sub.6 is an amino acid sequence containing 3 to 5 amino acid residues; (g) A.sub.7 is an amino acid residue; (h) A.sub.8 is an amino acid sequence containing 10 to 12 amino acid residues; and (i) A.sub.9 is an amino acid sequence containing 5 to 6 amino acid residues. Especially preferred NAP domains include those wherein A.sub.2 is an amino acid sequence containing 4 to 5 amino acid residues and A.sub.4 is an amino acid sequence containing 6 to 16 amino acid residues. More preferred are NAP domains, wherein: (a) A.sub.1 has Glu as its fourth amino acid residue; (b) A.sub.2 has Gly as its first amino acid residue; (c) A.sub.8 has Gly as its third amino acid residue and Arg as its sixth amino acid residue; and (d) A.sub.9 has Val as its first amino acid residue. More preferably, A.sub.3 has Asp or Glu as its first amino acid residue and Lys or Arg as its third amino acid residue and A.sub.7 is Val or Gln. Also, more preferably A.sub.8 has Leu or Phe as its fourth amino acid residue and Lys or Tyr as its fifth amino acid residue. Also preferred are NAP domains where, when A.sub.8 has 11 or 12 amino acid residues, Asp or Gly is its penultimate amino acid residue, and, where when A.sub.8 has 10 amino acids, Gly is its tenth amino acid residue. For expression of recombinant protein in certain expression systems, a recombinant NAP may additionally include an amino acid sequence for an appropriate secretion signal. Certain representative NAP domains include the sequences depicted in FIG. 11, particularly the sequences between (and including) the cysteines designated as Cysteine 1 and Cysteine 10.
The isolated NAPs of the present invention include those having variations in the disclosed amino acid sequence or sequences, including fragments, naturally occurring mutations, allelic variants, randomly generated artificial mutants and intentional sequence variations, all of which conserve anticoagulant activity. The term "fragments" refers to any part of the sequence which contains fewer amino acids than the complete protein, as for example, partial sequences excluding portions at the amino-terminus, carboxy-terminus or between the aminoterminus and carboxy-terminus of the complete protein.
The isolated NAPs of the present invention also include proteins having a recombinant amino acid sequence or sequences which conserve the anticoagulant activity of the NAP domain amino acid sequence or sequences. These recombinant proteins include hybrid proteins, such as fusion proteins, proteins resulting from the expression of multiple genes within the expression vector, proteins resulting from expression of multiple genes within the chromosome of the host cell, and may include a polypeptide having anticoagulant activity of a disclosed protein linked by peptide bonds to a second polypeptide. The recombinant proteins also include variants of the NAP domain amino acid sequence or sequences of the present invention that differ only by conservative amino acid substitution. Conservative amino acid substitutions are defined as "sets" in Table I of Taylor, W. R., J. Mol. Biol., 188:233 (1986). The recombinant proteins also include variants of the disclosed isolated NAP domain amino acid sequence or sequences of the present invention in which amino acid substitutions or deletions are made which conserve the anticoagulant activity of the isolated NAP domain sequence or sequences.
One preferred embodiment of the present invention is a protein isolated by biochemical methods from the nematode, Ancylostoma caninum, as described in Example 1. This protein increases the clotting time of human plasma in the PT and aPTT assays, contains one NAP domain, and is characterized by an N-terminus having the amino acid sequence, Lys-Ala-Tyr-Pro-Glu-Cys-Gly-Glu-Asn-Glu-Trp-Leu-Asp �SEQ. ID. NO. 6!, and a molecular weight of about 8.7 kilodaltons to about 8.8 kilodaltons as determined by mass spectrometry.
Further preferred embodiments of the present invention include the proteins having anticoagulant activity made by recombinant methods from the cDNA library isolated from the nematode, Ancylostoma caninum, for example, NAP isoform 5, NAP isoform 6, Pro-NAP isoform 5 and Pro-NAP isoform 6, the amino acids sequences of which are shown in FIG. 2, FIG. 4, FIG. 5 and FIG. 6, respectively. Each such preferred embodiment increases the clotting time of human plasma in the PT and aPTT assays and contains one NAP domain. Each is further characterized by an amino acid sequence which includes the amino acid sequence, Lys-Ala-Tyr-Pro-Glu-Cys-Gly-Glu-Asn-Glu-Trp-Leu-Asp �SEQ. ID. NO. 6!.
With respect to "isolated proteins", the proteins of the present invention are isolated by methods of protein purification well known in the art, or as disclosed below. They may be isolated from a natural source, from a chemical mixture after chemical synthesis on a solid phase or in solution such as solid-phase automated peptide synthesis, or from a cell culture after production by recombinant methods.
1. NAP Isolated From Natural Sources
The preferred isolated proteins (NAPs) of the present invention may be isolated and purified from natural sources. Preferred as natural sources are nematodes; suitable nematodes include intestinal nematodes such as Ancylostoma caninum, Ancylostoma ceylanicum, Ancylostoma duodenale, Necator americanus and Heligmosomoides polygyrus. Especially preferred as a natural source is the nematode, the dog hookworm, Ancylostoma caninum.
The preferred proteins of the present invention are isolated and purified from their natural sources by methods known in the biochemical arts. These methods include preparing a soluble extract and enriching the extract using chromatographic methods on different solid support matrices. Preferred methods of purification would include preparation of a soluble extract of a nematode in 0.02M Tris-HCl, pH 7.4 buffer containing various protease inhibitors, followed by sequential chromatography of the extract through columns containing Concanvalin-A Sepharose matrix, Poros20 HQ cation-ion exchange matrix, Superdex30 gel filtration matrix and a C18 reverse-phase matrix. The fractions collected from such chromatography columns may be selected by their ability to increase the clotting time of human plasma, as measured by the PT and aPTT assays, or their ability to inhibit factor Xa amidolytic activity as measured in a calorimetric amidolytic assay using purified enzyme. An example of a preferred method of purification of an isolated protein of the present invention would include that as disclosed in Example 1.
The preferred proteins of the present invention, when purified from the natural source, Ancylostoma caninum, as described, include those which contain the amino acid sequence: Lys-Ala-Tyr-Pro-Glu-Cys-Gly-Glu-Asn-Glu-Trp-Leu-Asp �SEQ. ID. NO. 6!. Especially preferred are the purified proteins having this amino acid sequence at its amino terminus. More especially preferred are purified proteins having the amino acid sequences shown in FIG. 2 (NAP isoform 5) or FIG. 4 (NAP isoform 6). One preferred protein of the present invention was demonstrated to have the amino acid sequence, Lys-Ala-Tyr-Pro-Glu-Cys-Gly-Glu-Asn-Glu-Trp-Leu-Asp �SEQ. ID. NO. 6! at its amino-terminus and a molecular weight of 8.7 to 8.8 kilodaltons, as determined by mass spectrometry.
2. NAP Made by Chemical Synthesis
The preferred isolated NAPs of the present invention may be synthesized by standard methods known in the chemical arts.
The isolated proteins of the present invention may be prepared using solid-phase synthesis, such as that described by Merrifield, J. Amer. Chem. Soc., 85:2149 (1964) or other equivalent methods known in the chemical arts, such as the method described by Houghten in Proc. Natl. Acad. Sci., 82:5132 (1985).
Solid-phase synthesis is commenced from the C-terminus of the peptide by coupling a protected amino acid or peptide to a suitable insoluble resin. Suitable resins include those containing chloromethyl, bromomethyl, hydroxylmethyl, aminomethyl, benzhydryl, and t-alkyloxycarbonylhydrazide groups to which the amino acid can be directly coupled.
In this solid phase synthesis, the carboxy terminal amino acid, having its alpha amino group and, if necessary, its reactive side chain group suitably protected, is first coupled to the insoluble resin. After removal of the alpha amino protecting group, such as by treatment with trifluoroacetic acid in a suitable solvent, the next amino acid or peptide, also having its alpha amino group and, if necessary, any reactive side chain group or groups suitably protected, is coupled to the free alpha amino group of the amino acid coupled to the resin. Additional suitably protected amino acids or peptides are coupled in the same manner to the growing peptide chain until the desired amino acid sequence is achieved. The synthesis may be done manually, by using automated peptide synthesizers, or by a combination of these.
The coupling of the suitably protected amino acid or peptide to the free alpha amino group of the resin-bound amino acid can be carried out according to conventional coupling methods, such as the azide method, mixed anhydride method, DCC (dicyclohexylcarbodiimide) method, activated ester method (p-nitrophenyl ester or N-hydroxysuccinimide ester), BOP (benzotriazole-1-yloxy-tris (diamino) phosphonium hexafluorophosphate) method or Woodward reagent K method.
It is common in peptide synthesis that the protecting groups for the alpha amino group of the amino acids or peptides coupled to the growing peptide chain attached to the insoluble resin will be removed under conditions which do not remove the side chain protecting groups. Upon completion of the synthesis, it is also common that the peptide is removed from the insoluble resin, and during or after such removal, the side chain protecting groups are removed.
Suitable protecting groups for the alpha amino group of all amino acids and the omega amino group of lysine include benzyloxycarbonyl, isonicotinyloxycarbonyl, o-chlorobenzyloxycarbonyl, p-nitrophenyloxycarbonyl, p-methoxyphenyloxycarbonyl, t-butoxycarbonyl, tamyloxycarbonyl, adamantyloxycarbonyl, 2-(4-biphenyl)-2-propyloxycarbonyl, 9-fluorenylmethoxycarbonyl, methylsulfonylethoxylcarbonyl, trifluroacetyl, phthalyl, formyl, 2-nitrophenylsulfphenyl, diphenylphosphinothioyl, dimethylphosphinothioyl, and the like.
Suitable protecting groups for the carboxy group of aspartic acid and glutamic acid include benzyl ester, cyclohexyl ester, 4-nitrobenzyl ester, t-butyl ester, 4-pyridylmethyl ester, and the like.
Suitable protecting groups for the guanidino group of arginine include nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzenesulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl, 1,3,5-trimethylphenylsulfonyl, and the like.
Suitable protecting groups for the thiol group of cysteine include p-methoxybenzyl, triphenylmethyl, acetylaminomethyl, ethylcarbamoyl, 4-methylbenzyl, 2,4,6-trimethylbenzyl, and the like.
Suitable protecting groups for the hydroxy group of serine include benzyl, t-butyl, acetyl, tetrahydropyranyl, and the like.
The completed peptide may be cleaved from the resin by treatment with liquid hydrofluoric acid containing one or more thio-containing scavengers at reduced temperatures. The cleavage of the peptide from the resin by such treatment will also remove all side chain protecting groups from the peptide.
The cleaved peptide is dissolved in dilute acetic acid followed by filtration, then is allowed to refold and establish proper disulfide bond formation by dilution to a peptide concentration of about 0.5 mM to about 2 mM in a 0.1M acetic acid solution. The pH of this solution is adjusted to about 8.0 using ammonium hydroxide and the solution is stirred open to air for about 24 to about 72 hours.
The refolded peptide is purified by chromatography, preferably by high pressure liquid chromatography on a reverse phase column, eluting with gradient of acetonitrile in water (also containing 0.1% trifluoroacetic acid), with the preferred gradient running from 0 to about 80% acetonitrile in water. Upon collection of fractions containing the pure peptide, the fractions are pooled and lyophilized to the solid peptide.
3. NAP Made By Recombinant Methods
Alternatively, the preferred isolated NAPs the present invention may be made by recombinant DNA methods well known in the biological arts. Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning, A Laboratory Manual, Second Edition, volumes 1 to 3, Cold Spring Harbor Laboratory Press (1989).
Recombinant DNA methods allow segments of genetic information, DNA, from different organisms, to be joined together outside of the organisms from which the DNA was obtained and this hybrid DNA to be incorporated into a cell that will allow the production of the protein for which the original DNA encodes.
Genetic information encoding a protein of the present invention may be obtained from the genomic DNA or mRNA of an organism by methods well known in the art. Preferred methods of obtaining this genetic information include isolating mRNA from an organism, converting it to its complementary DNA (cDNA), incorporating the cDNA into an appropriate cloning vector, and identifying the clone which contains the recombinant cDNA encoding the desired protein by means of hybridization with appropriate oligonucleotide probes constructed from known sequences of the protein.
The genetic information in the recombinant cDNA encoding a protein of the present invention may be ligated into an expression vector, the vector introduced into host cells, and the genetic information expressed as the protein encoded for.
(A) Preparation of cDNA Library.
Preferred natural sources of mRNA from which to construct a cDNA library are nematodes which include intestinal nematodes such as Ancylostoma caninum, Ancylostoma ceylanicum, Ancylostoma duodenale, Necator americanus and Heligmosomoides polygyrus. Especially preferred as a natural source of mRNA is the nematode, Ancylostoma caninum.
Preferred methods of isolating mRNA encoding a protein of the present invention, along with other mRNA, from an organism include chromatography on poly U or poly T affinity gels. Especially preferred methods of isolating the mRNA from nematodes include the procedure and materials provided in the QuickPrep mRNA Purification kit (Pharmacia).
Preferred methods of obtaining double-stranded cDNA from isolated mRNA include synthesizing a single-stranded cDNA on the mRNA template using a reverse transcriptase, degrading the RNA hybridized to the cDNA strand using a ribonuclease (RNase), and synthesizing a complementary DNA strand by using a DNA polymerase to give a double-stranded cDNA. Especially preferred methods include those wherein about 3 micrograms of mRNA isolated from a nematode is converted into double-stranded cDNA making use of Avian Myeloblastosis Virus reverse transcriptase, RNase H, and E. coli DNA polymerase and T4 DNA polymerase.
cDNA encoding a protein of the present invention, along with the other cDNA in the library constructed as above, are then ligated into cloning vectors. Cloning vectors include a DNA sequence which accommodates the cDNA from the cDNA library. The vectors containing the cDNA library are introduced into host cells that can exist in a stable manner and provide a environment in which the cloning vector is replicated. Suitable cloning vectors include plasmids, bacteriophages, viruses and cosmids. Preferred cloning vectors include the bacteriophages. Cloning vectors which are especially preferred include the bacteriophage, lambda gt11 Sfi-Not vector.
The construction of suitable cloning vectors containing the cDNA library and control sequences employs standard ligation and restriction techniques which are well known in the art. Isolated plasmids, DNA sequences or synthesized oligonucleotides are cleaved, tailored and religated in the form desired.
With respect to restriction techniques, site-specific cleavage of cDNA is performed by treating with suitable restriction enzyme under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. For example, see the product catalogs of New England Biolabs, Promega and Stratagene Cloning Systems.
Generally, about 1 microgram of the cDNA is cleaved by treatment in about one unit of a restriction enzyme in about 20 microliters of buffer solution. Typically, an excess of restriction enzyme is used to ensure complete cleavage of the cDNA. Incubation times of about 1 to 2 hours at about 37.degree. C. are usually used, though exceptions are known. After each cleavage reaction, the protein may be removed by extraction with phenol/chloroform, optionally followed by chromatography over a gel filtration column, such as Sephadex.RTM. G50. Alternatively, cleaved cDNA fragments may be separated by their sizes by electrophoresis in polyacrylamide or agarose gels and isolated using standard techniques. A general description of size separations is found in methods of Enzymology, 65:499-560 (1980).
The restriction enzyme-cleaved cDNA fragments are then ligated into a cloning vector.
With respect to ligation techniques, blunt-end legations are usually performed in about 15 to about 30 microliters of a pH 7.5 buffer comprising about 1 mM ATP and about 0.3 to 0.6 (Weiss) units of T4 DNA ligase at about 14.degree. C. Intermolecular "sticky end" legations are usually performed at about 5 to 100 nanomolar total-end DNA concentrations. Intermolecular blunt-end ligations (usually employing about 10 to 30-fold molar excess of linkers) are performed at about 1 micromolar total-end DNA concentrations.
(B) Preparation of cDNA Encoding NAP.
Cloning vectors containing the cDNA library prepared as disclosed are introduced into host cells, the host cells are cultured, plated, and then probed with a hybridization probe to identify clones which contain the recombinant cDNA encoding a protein of the present invention. Preferred host cells include bacteria when phage cloning vectors are used. Especially preferred host cells include E. coli strains such as strain Y1090.
Alternatively, the recombinant cDNA encoding a protein of the present invention may be obtained by expression of such protein on the outer surface of a filamentous phage and then isolating such phage by binding them to a target protein involved in blood coagulation.
An important and well known feature of the genetic code is its redundancy--more than one triplet nucleotide sequence codes for one amino acid. Thus, a number of different nucleotide sequences are possible for recombinant cDNA molecules which encode a particular amino acid sequence for a NAP of the present invention. Such nucleotide sequences are considered functionally equivalent since they can result in the production of the same amino acid sequence in all organisms. Occasionally, a methylated variant of a purine or pyrimidine may be incorporated into a given nucleotide sequence. However, such methylations do not affect the coding relationship in any way.
(1) Using Oligonucleotide Probes.
Hybridization probes and primers are oligonucleotide sequences which are complementary to all or part of the recombinant cDNA molecule that is desired. They may be prepared using any suitable method, for example, the phosphotriester and phosphodiester methods, described respectively in Narang, S. A. et al., Methods in Enzymology, 68:90 (1979) and Brown, E. L. et al., Methods in Enzymology, 68:109 (1979), or automated embodiments thereof. In one such embodiment, diethylphosphoramidites are used as starting materials and may be synthesized as described by Beaucage et al, Tetrahedron Letters, 22:1859-1862 (1981). One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Pat. No. 4,458,066. Probes differ from primers in that they are labelled with an enzyme, such as horseradish peroxidase, or radioactive atom, such as .sup.32 p, to facilitate their detection. A synthesized probe is radiolabeled by nick translation using E. coli DNA polymerase I or by end labeling using alkaline phosphatase and T4 bacteriophage polynucleotide kinase.
Preferred hybridization probes include oligonucleotide sequences which are complementary to a stretch of the single-stranded cDNA encoding a portion of the amino acid sequence of NAP purified from the nematode, the dog hookworm, Ancylostoma caninum. For example, a portion of the amino acid sequence shown in FIG. 2 (NAP isoform 5) or FIG. 4 (NAP isoform 6) can be used. Especially preferred hybridization probes include those wherein their oligonucleotide sequence is complementary to the stretch of the single-stranded cDNA encoding the amino acid sequence: Lys-Ala-Tyr-Pro-Glu-Cys-Gly-Glu-Asn-Glu-Trp �SEQ. ID. NO. 7!. Such hybridization probes include the degenerate probe having the oligonucleotide sequence: AAR GCi TAY CCi GAR TGY GGi GAR AAY GAR TGG �SEQ. ID. NO. 1!, wherein R is A or G, Y is T or C, and i is inosine. A preferred recombinant cDNA molecule encoding a protein of the present invention is identified by its ability to hybridize to this probe.
Upon identification of the clone containing the desired cDNA, amplification is used to produce large quantities of a gene encoding a protein of the present invention in the form of a recombinant cDNA molecule.
Preferred methods of amplification include the use of the polymerase chain reaction (PCR). See, e.g., PCR Technology, W. H. Freeman and Company, New York (Edit. Erlich, H. A. 1992). PCR is an in vitro amplification method for the synthesis of specific DNA sequences. In PCR, two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the cDNA of the clone are used. A repetitive series of cycles involving cDNA denaturation into single strands, primer annealing to the single-stranded cDNA, and the extension of the annealed primers by DNA polymerase results in number of copies of cDNA, whose termini are defined by the 5-ends of the primers, approximately doubling at every cycle. Ibid., p.1. Through PCR amplification, the coding domain and any additional primer encoded information such as restriction sites or translational signals (signal sequences, start codons and/or stop codons) of the recombinant cDNA molecule to be isolated is obtained.
Preferred conditions for amplification of cDNA include those using Taq polymerase and involving 30 temperature cycles of: 1 minute at 95.degree. C.; 1 minute at 50.degree. C.; 1.5 minutes at 72.degree. C. Preferred primers include the oligo(dT)-NotI primer, AATTCGCGGC CGC(T) .sub.15 �SEQ. ID. NO. 8!, obtained from Promega Corp. in combination with either (i) the degenerate primer having the oligonucleotide sequence: AAR GCi TAY CCi GAR TGY GGi GAR AAY GAR TGG �SEQ. ID. NO. 1!, wherein R is A or G, Y is T or C, and i is inosine, or (ii) the lambda gt11 primer #1218, GGTGGCGACG ACTCCTGGAG CCCG �SEQ. ID. NO. 9!, obtained from New England Biolabs.
The nucleic acid sequence of a recombinant cDNA molecule made as disclosed is determined by the dideoxy method of Sanger, F. et al., Proc. Natl. Acad. Sci. USA, 74:5463 (1977) as further described by Messing, et al., Nucleic Acids Res., 9:309 (1981).
Preferred recombinant cDNA molecules made as disclosed include those having the nucleic acid sequences of FIG. 1 (NAP5 gene) and FIG. 3 (NAP6 gene).
(2) Using NAP cDNAs As Probes.
Preferred recombinant cDNA molecules made by the above method, but using as hybridization probes fragments derived from the NAP5 gene (FIG. 1) and the NAP6 gene (FIG. 2), include those having the nucleic acid sequences of FIGS. 7A, 7B, 7C, 7D, 7E or 7F.
Also especially preferred as hybridization probes are oligonucleotide sequences encoding substantially all of the amino acid sequence of NAP purified from the nematode, the dog hookworm, Ancylostoma caninum. Especially preferred probes include those having the nucleic acid sequences shown in FIG. 1 (NAP5 gene): AAG GCA TAC CCG GAG TGT GGT GAG AAT GAA TGG CTC GAC GAC TGT GGA ACT CAG AAG CCA TGC GAG GCC AAG TGC AAT GAG GAA CCC CCT GAG GAG GAA GAT CCG ATA TGC CGC TCA CGT GGT TGT TTA TTA CCT CCT GCT TGC GTA TGC AAA GAC GGA TTC TAC AGA GAC ACG GTG ATC GGC GAC TGT GTT AGG GAA GAA GAA TGC GAC CAA CAT GAG ATT ATA CAT GTC TGA �SEQ. ID. NO. 2!, or FIG. 3 (NAP6 gene): AAG GCA TAC CCG GAG TGT GGT GAG AAT GAA TGG CTC GAC GTC TGT GGA ACT AAG AAG CCA TGC GAG GCC AAG TGC AGT GAG GAA GAG GAG GAA GAT CCG ATA TGC CGA TCA TTT TCT TGT CCG GGT CCC GCT GCT TGC GTA TGC GAA GAC GGA TTC TAC AGA GAC ACG GTG ATC GGC GAC TGT GTT AAG GAA GAA GAA TGC GAC CAA CAT GAG ATT ATA CAT GTC TGA �SEQ. ID. NO. 3!.
(3) Using Phage Display.
Disclosed herein is a method to select cDNAs encoding the proteins of the present invention from whole cDNA libraries making use of filamentous phage display technology. Current display technology with filamentous phage relies on the in-frame insertion of coding regions of interest into gene 3 or gene 8 which code for the attachment protein and major coat protein of the phage, respectively. Those skilled in the art will recognize that various difficulties are inherent in performing this with a vast mixture of cDNAs of unknown sequence and that the most practical way to obtain functional display of cDNA products would consist of fusing the cDNAs through their 5'-end. Indeed, cDNA libraries of sufficient size may contain several cDNAs which derive from the same mRNA but which are 5'-terminally truncated at various positions such that some of them may be expressed as fusion products. A strategy along this line, which relies on the ability of the leucine zippers Jun and Fos to form heterodimers was recently described. See, Crameri, R. and Suter, M., Gene, 137:69-75 (1993).
We have found a novel alternative and direct way to convalently link cDNA gene products to the phage surface; the finding is based on the observation that proteins fused to the C-terminus of phage coat protein 6 can be functionally displayed. This observation has led to the development of a phagemid system as described herein which allows the expression of functionally displayed cDNA products, which in turn permits the affinity-selection of phage particles which contain the cDNA required for the production of the displayed cDNA product. This system provides the basis for the isolation of cDNAs which encode a protein of the present invention. Once isolated, recombinant cDNA molecules containing such cDNA can be used for expression of the proteins of the present invention in other expression systems. The recombinant cDNA molecules made in this way are considered to be within the scope of the present invention.
Recombinant cDNA molecules of the present invention are isolated by preparing a cDNA library from a natural source (as for example, a nematode such as a hookworm), ligating this cDNA library into appropriate phagemid vectors, transforming host cells with these vectors containing the cDNAs, culturing the host cells, infecting the transformed cells with an appropriate helper phage, separating phage from the host cell culture, separating phage expressing a protein of the present invention on its surface, isolating these phage, and isolating a recombinant cDNA molecule from such phage.
The phagemid vectors are constructed using the pUC119 expression vector described by Vieira, J. and Messing, J., Methods in Enzymology, 153:3-11 (1987). The filamentous phage gene 6 encoding a surface protein of the phage is modified on its 5' and 3' ends by the addition of HindIII and SfiI restriction sites, respectively, by use of three forward primers and one backward primer using PCR. This results in three DNA fragments which are further modified by addition to their 3' ends of NotI and BamHI restriction sites by PCR. After separate digestion of the three DNA fragments with HindIII and BamHI, the three DNA fragments are ligated into the pUC119 to give pDONG61, pDONG62 and pDONG63 expression vectors. These vectors permit the insertion of cDNA as SfiI-NotI fragments into them.
cDNA libraries are prepared from natural sources, such as nematodes, as described in Example 2. Preferred nematodes from which to make such libraries include the intestinal nematodes such as Ancylostoma caninum, Ancylostoma ceylanicum, Ancylostoma duodenale, Necator arnericanus and Heligmosomoides polygyrus.
A cDNA library as SfiI-NotI fragments may be directly directionally ligated into the phagemid vectors pDONG61, pDONG62 and pDONG63. Alternatively, a cDNA library which has been ligated into the lambda gt11 phage vector as described in Example 2 can be recovered by PCR, followed by isolation with electrophoresis and then directional ligation into these vectors. In the latter approach, preferred conditions for PCR use Taq polymerase; the primers, lambda gt11 primer #1218 having the sequence GGTGGCGACG ACTCCTGGAG CCCG �SEQ. ID. NO. 9! (New England Biolabs, Beverly, Mass., USA) and the oligo(dT)-NotI primer having the sequence, AATTCGCGGC CGC(T) .sub.15 �SEQ. ID. NO. 8! (Promega Corp.); and 20 temperature cycles of 1 minute at 95.degree. C., 1 minute at 50.degree. C., and 3 minutes at 72.degree. C., followed by 10 minutes at 65.degree. C.
Host cells are transformed with the pDONG expression vectors containing a cDNA library. Preferred host cells include E. coli strains, with strain TG1 being especially preferred. Preferred methods for the transformation of E. coli host cells include electroporation.
The transformed cells are cultured at 37.degree. C. in LB medium supplemented with 1% glucose and 100 micrograms/ml carbenicillin until the optical absorbance at 600 nm reaches the value of 0.5 and then are infected with VCSM13 helper phage (Stratagene) at a multiplicity of infection (moi) of 20.
The phage are separated from the culture by centrifugation, then are purified by precipitations with polyethylene glycol/sodium chloride.
The phage which express a NAP of the present invention on their surface are isolated by taking advantage of the ability of the NAP to bind to a target protein involved in blood coagulation, for example, Factor Xa.
Preferred methods of isolating such phage include a method comprising the steps of:
(1) combining a solution of factor Xa labelled to biotin with a solution of such phage;
(2) incubating this mixture;
(3) contacting a solid phase labelled with streptavidin with this mixture;
(4) incubating the solid phase with the mixture;
(5) removing the solid phase from the mixture and contacting the solid phase with buffer to remove unbound phage;
(6) contacting the solid phase with a second buffer to remove the bound phage from the solid phase;
(7) isolating such phage;
(8) transforming host cells with such phage;
(9) culturing the transformed host cells;
(10) infecting transformed host cells with VCSM13 helper phage;
(11) isolating the phage from the host cell culture; and
(12) repeating steps (1) to (11) four more times.
An especially preferred method of isolating such phage include the method as detailed in Example 10. Single-stranded DNA was prepared from the isolated phages and their inserts 3' to the filamentous phage gene 6 sequenced.
FIG. 9 depicts the recombinant cDNA molecule, NAPc2 �SEQ. ID. NO. 47!, isolated by the phage display method. The deduced amino acid sequence of the protein of the present invention encoded by NAPc2 is also shown in this figure.
(C) Preparation of Recombinant NAP.
The recombinant cDNA molecules of the present invention when isolated as disclosed are used to obtain expression of the NAPs of the present invention. Generally, a recombinant cDNA molecule of the present invention is incorporated into an expression vector, this expression vector is introduced into an appropriate host cell, the host cell is cultured, and the expressed protein is isolated.
Expression vectors are DNA sequences that are required for the transcription of cloned copies of genes and translation of their mRNAs in an appropriate host. These vectors can express either procaryotic or eucaryotic genes in a variety of cells such as bacteria, yeast, mammalian, plant and insect cells. Proteins may also be expressed in a number of virus systems.
Suitably constructed expression vectors contain an origin of replication for autonomous replication in host cells, or are capable of integrating into the host cell chromosomes. Such vectors will also contain selective markers, a limited number of useful restriction enzyme sites, a high copy number, and strong promoters. Promoters are DNA sequences that direct RNA polymerase to bind to DNA and initiate RNA synthesis; strong promoters cause such initiation at high frequency. The preferred expression vectors of the present invention are operatively linked to a recombinant cDNA molecule of the present invention, i.e., the vectors are capable directing both replication of the attached recombinant cDNA molecule and expression of the protein encoded by the recombinant cDNA molecule. Expression vectors may include, but are not limited to cloning vectors, modified cloning vectors and specifically designed plasmids or viruses.
Suitable host cells for expression of the proteins of the present invention include bacteria, yeast, mammalian, plant and insect cells. With each type of cell and species therein certain expression vectors are appropriate as will be disclosed below.
Procaryotes may be used for expression of the proteins of the present invention. Suitable bacteria host cells include the various strains of E. coli, Bacillus subtilis, and various species of Pseudomonas. In these systems, plasmid vectors which contain replication sites and control sequences derived from species compatible with the host are used. Suitable vectors for E. coli are derivatives of pBR322, a plasmid derived from an E. coli species by Bolivar et al., Gene, 2:95 (1977). Common procaryotic control sequences, which are defined herein to include promoters for transcription, initiation, optionally with an operator, along with ribosome binding site sequences, include the beta-lactamase and lactose promoter systems (Chang et al., Nature, 198:1056 (1977)), the tryptophan promoter system (Goeddel et al., Nucleic Acids Res., 8:4057 (1980)) and the lambda-derived-P.sub.L promoter and N-gene ribosome binding site (Shimatake et al., Nature, 292:128 (1981)). However, any available promoter system compatible with procaryotes can be used. Preferred procaryote expression systems include E. coli and their expression vectors.
Eucaryotes may be used for expression of the proteins of the present invention. Eucaryotes are usually represented by the yeast and mammalian cells. Suitable yeast host cells include Saccharomyces cerevisiae and Pichia pastoris. Suitable mammalian host cells include COS and CHO (chinese hamster ovary) cells.
Expression vectors for the eucaryotes are comprised of promoters derived from appropriate eucaryotic genes. Suitable promoters for yeast cell expression vectors, include promoters for synthesis of glycolytic enzymes, including those for 3-phosphoglycerate kinase gene in Saccharomyces cerevisiae (Hitzman et al., J. Biol. Chem., 255:2073 (1980)) and those for the metabolism of methanol as the alcohol oxidase gene in Pichia pastoris (Stroman et al., U.S. Pat. Nos. 4,808,537 and 4,855,231). Other suitable promoters include those from the enolase gene (Holland, M. J. et al., J. Biol. Chem., 256:1385 (1981)) or the Leu2 gene obtained from YEpl3 (Broach, J. et al., Gene, 8:121 (1978)). Preferred yeast expression systems include Pichia pastoris and their expression vectors.
Suitable promoters for mammalian cell expression vectors include the early and late promoters from SV40 (Fiers, et al., Nature, 273:113 (1978)) or other viral promoters such as those derived from polyoma, adenovirus II, bovine papilloma virus or avian sarcoma viruses. Suitable viral and mammalian enhancers may also be incorporated into these expression vectors.
Suitable promoters for plant cell expression vectors include the nopaline synthesis promoter described by Depicker, A. et al., Mol. Appl. Gen., 1:561 (1978).
Suitable promoters for insect cell expression vectors include modified versions of the system described by Smith et al., U.S. Pat. No. 4,745,051. The expression vector comprises a baculovirus polyhedrin promoter under whose control a cDNA molecule encoding a protein can be placed.
Host cells are transformed by introduction of expression vectors of the present invention into them. Transformation is done using standard techniques appropriate for each type of cell. The calcium treatment employing calcium chloride described in Cohen, S. N., Proc. Natl. Acad. Sci. USA, 69:2110 (1972), or the RbCl method described in Maniatis et al., Molecular Cloning: A Laboratory Manual, p. 254, Cold Spring Harbor Press (1982) is used for procaryotes or other cells which contain substantial cell wall barriers. The transformation of yeast is carried out as described in Van Solingen, P. et al., J. Bacter., 130:946 (1977) and Hsiao, C. L. et al., Proc. Natl. Acad. Sci. USA, 76:3829 (1979). Mammalian cells without much cell wall are transformed using the calcium phosphate procedure of Graham and van der Eb, Virology, 52:546 (1978). Plant cells are transformed by infection with Agrobacterium tumefaciens as described in Shaw, C. et al., Gene, 23:315 (1983). Preferred methods of transforming E. coli and Pichia pastoris with expression vectors include electroporation.
Transformed host cells are cultured under conditions, such as type of media, temperature, oxygen content, fluid motion, etc., well known in the biological arts.
The recombinant proteins of the present invention are isolated from the host cell or media by standard methods well known in the biochemical arts, which include the use chromatography methods. Preferred methods of purification would include sequential chromatography of an extract through columns containing Poros20 HQ anion-ion exchange matrix or Poros20 HS cation exchange matrix, Superdex30 gel filtration matrix and a C18 reverse-phase matrix. The fractions collected after one such chromatography column may be selected by their ability to increase the clotting time of human plasma, as measured by the PT and aPTT assays, or their ability to inhibit factor Xa amidolytic activity as measured in a colorimetric assay. Examples of a preferred methods of purification of a recombinant protein of the present invention are disclosed in Examples 3, 5 and 6.
4. Methods of Using NAP.
In one aspect, the present invention includes methods of collecting mammalian plasma, comprising adding to a blood collection tube an amount of a protein of the present invention sufficient to inhibit the formation of a clot when mammalian blood is drawn into the tube, adding mammalian blood to said tube, separating the red blood cells from the mammalian plasma, and collecting the mammalian plasma.
Blood collection tubes include stoppered test tubes having a vacuum therein as a means to draw blood obtained by venipuncture into the tubes. Preferred test tubes include those which are made of borosilicate glass, and have the dimensions of, for example, 10.25.times.47 mm, 10.25.times.50 mm, 10.25.times.64 mm, 10.25.times.82 mm, 13.times.75 mm, 13.times.100 mm, 16.times.75 mm, 16.times.100 mm or 16.times.125 mm. Preferred stoppers include those which can be easily punctured by a blood collection needle and which when placed onto the test tube provide a seal sufficient to prevent leaking of air into the tube.
The proteins of the present invention are added to the blood collection tubes in variety of forms well known in the art, such as a liquid composition thereof, a solid composition thereof, or a liquid composition which is lyophilized to a solid in the tube. The amount added to such tubes is that amount sufficient to inhibit the formation of a clot when mammalian blood is drawn into the tube. The proteins of the present invention are added to blood collection tubes in such amounts that, when combined with 2 to 10 ml of mammalian blood, the concentration of such proteins will be sufficient to inhibit clot formation. Typically, this effective concentration will be about 1 to 10,000 nM, with 10 to 1000 nM being preferred. Alternatively, the proteins of the present invention may be added to such tubes in combination with other clot-inhibiting additives, such as heparin salts, EDTA salts, citrate salts or oxalate salts.
After mammalian blood is drawn into a blood collection tube containing either a protein of the present invention or the same in combination with other clot-inhibiting additives, the red blood cells are separated from the mammalian plasma by centrifugation. The centrifugation is performed at g-forces, temperatures and times well known in the medical arts. Typical conditions for separating plasma from red blood cells include centrifugation at a centrifugal force of about 100.times.g to about 1500.times.g, at a temperatures of about 5 to about 25.degree. C., and for a time of about 10 to about 60 minutes.
The mammalian plasma may be collected by pouring it off into a separate container, by withdrawing it into a pipette or by other means well known to those skilled in the medical arts.
In another aspect, the present invention includes methods for preventing thrombosis (clot formation) in a mammal, comprising administering to said mammal a therapeutically effective amount of a protein or a pharmaceutical composition of the present invention.
The proteins or pharmaceutical compositions of the present invention are administered in vivo, ordinarily in a mammal, preferably in a human. In employing them in vivo, the proteins or pharmaceutical compositions can be administered to a mammal in a variety of ways, including orally, parenterally, intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperitoneally, employing a variety of dosage forms. Administration is preferably parenteral, such as intravenous on a daily basis. Alternatively, administration is preferably oral, such as by tablets, capsules or elixers taken on a daily basis.
In practicing the methods of the present invention, the proteins or pharmaceutical compositions of the present invention are administered alone or in combination with one another, or in combination with other therapeutic or in vivo diagnostic agents.
As is apparent to one skilled in the medical art, a therapeutically effective amount of the proteins or pharmaceutical compositions of the present invention will vary depending upon the age, weight and mammalian species treated, the particular proteins employed, the particular mode of administration and the desired affects and the therapeutic indication. Because these factors and their relationship to determining this amount are well known in the medical arts, the determination of therapeutically effective dosage levels, the amount necessary to achieve the desired result of preventing thrombosis, will be within the ambit of one skilled in these arts.
Typically, administration of the proteins or pharmaceutical composition of the present invention is commenced at lower dosage levels, with dosage levels being increased until the desired effect of preventing in vivo thrombosis is achieved which would define a therapeutically effective amount. For the proteins of the present invention, alone or as part of a pharmaceutical composition, such doses are between about 0.01 mg/kg and 100 mg/kg body weight, preferably between about 0.01 and 10 mg/kg, body weight.
5. Utility.
Proteins of the present invention when made and selected as disclosed are useful as potent inhibitors of blood coagulation in vitro and in vivo. As such, these proteins are useful as in vitro diagnostic reagents to prevent the clotting of blood and are also useful as in vivo pharmaceutical agents to prevent thrombosis in mammals.
The proteins of the present invention are useful as in vitro diagnostic reagents for inhibiting clotting in blood drawing tubes. The use of stoppered test tubes having a vacuum therein as a means to draw blood obtained by venipuncture into the tube is well known in the medical arts. Kasten, B. L., "Specimen Collection", Laboratory Test Handbook, 2nd Edition, Lexi-Comp Inc., Cleveland pp. 16-17 (Edits. Jacobs, D. S. et al. 1990). Such vacuum tubes may be free of clot-inhibiting additives, in which case, they are useful for the isolation of mammalian serum from the blood. They may alternatively contain clot-inhibiting additives (such as heparin salts, EDTA salts, citrate salts or oxalate salts), in which case, they are useful for the isolation of mammalian plasma from the blood. The proteins of the present invention are potent inhibitors of blood clotting and as such, can be incorporated into blood collection tubes to prevent clotting of the mammalian blood drawn into them.
The proteins of the present invention are used alone, in combination of other proteins of the present invention, or in combination with other known inhibitors of clotting, in the blood collection tubes, for example, with heparin salts, EDTA salts, citrate salts or oxalate salts.
The amount to be added to such tubes, or effective amount, is that amount sufficient to inhibit the formation of a blood clot when mammalian blood is drawn into the tube. The proteins of the present invention are added to blood collection tubes in such amounts that, when combined with 2 to 10 ml of mammalian blood, the concentration of such proteins will be sufficient to inhibit the formation of blood clots. Typically, this effective amount is that required to give a final concentration in the blood of about 1 to 10,000 nM, with 10 to 1000 nM being preferred.
The proteins of the present invention may also be used to prepare diagnostic compositions. In one embodiment, diagnostic compositions are prepared by dissolving the proteins of the present invention into diagnostically acceptable carriers, which carriers include phosphate buffered saline (0.01M sodium phosphate+0.15M sodium chloride, pH 7.2 or Tris buffered saline (0.05M Tris-HCl+0.15M sodium chloride, pH 8.0). In another embodiment, the proteins of the present invention may be blended with other solid diagnostically acceptable carriers by methods well known in the art to provide solid diagnostic compositions. These carriers include buffer salts.
The addition of the proteins of the present invention to blood collection tubes may be accomplished by methods well known in the art, which methods include introduction of a liquid diagnostic composition thereof, a solid diagnostic composition thereof, or a liquid diagnostic composition which is lyophilized in such tubes to a solid plug of a solid diagnostic composition.
The use of blood collection tubes containing the diagnostic compositions of the present invention comprises contacting a effective amount of such diagnostic composition with mammalian blood drawn into the tube. Typically, when a sample of 2 to 10 ml of mammalian blood is drawn into a blood collection tube and contacted with such diagnostic composition therein; the effective amount to be used will include those concentrations of the proteins formulated as a diagnostic composition which in the blood sample are sufficient to inhibit the formation of blood clots. Preferred effective concentrations would be about 1 to 10,000 nM, with 10 to 1000 nM being especially preferred.
According to an alternate aspect of our invention, the proteins of the present invention are also useful as pharmaceutical agents for preventing thrombosis in a mammals. This prevention of thrombosis includes the preventing of abnormal thrombosis.
Conditions characterized by abnormal thrombosis are well known in the medical arts and include those involving the arterial and venous vasculature of mammals. With respect to the coronary arterial vasculature, abnormal thrombosis (thrombus formation) characterizes the rupture of an established atherosclerotic plaque which is the major cause of acute myocardial infarction and unstable angina, and also characterizes the occlusive coronary thrombus formation resulting from either thrombolytic therapy or percutaneous transluminal coronary angioplasty (PTCA). With respect to the venous vasculature, abnormal thrombosis characterizes the condition observed in patients undergoing major surgery in the lower extremities or the abdominal area who often suffer from thrombus formation in the venous vasculature resulting in reduced blood flow to the affected extremity and a predisposition for pulmonary embolism. Abnormal thrombosis further characterizes disseminated intravascular coagulopathy which commonly occurs within both vascular systems during septic shock, certain viral infections and cancer, a condition wherein there is rapid consumption of coagulation factors and systemic coagulation which results in the formation of life-threatening thrombi occurring throughout the microvasculature leading to widespread organ failure.
The recombinant cDNA molecules encoding the proteins of the present invention are useful in one aspect for isolating other recombinant cDNA molecules which also encode the proteins of the present invention. In another aspect, they are useful for expression in host cells of the proteins of the present invention.
To assist in understanding, the present invention will now be be further illustrated by the following examples. These examples as they relate to this invention should not, of course, be construed as specifically limiting the invention and such variations of the invention, now known or later developed, which would be within the purview of one skilled in the art are considered to fall within the scope of the invention as described herein and hereinafter claimed.
EXAMPLES
Example 1
Isolation of Novel Anticoagulant Protein (NAP) from Ancylostoma caninum.
(A) Preparation of the Ancylostoma caniumum Lysate. Frozen canine hookworms, Ancylostoma caniumum, were obtained from Antibody Systems (Bedford, Tex.). Hookworms were stored at -80.degree. C. until used for homogenate.
Hookworms were liquid nitrogen frozen and ground in a mortar followed by a homogenization on ice in homogenization buffer using a PotterS homogenizer with a teflon piston (B. Braun Melsungen AG, Germany). The homogenization buffer contained: 0.02M Tris-HCl pH 7.4, 0.05M NaCl, 0.001M MgCl.sub.2, 0.001M CaCl.sub.2, 1.0.times.10.sup.-5 M E-64 protease inhibitor (Boehringer Mannheim, Germany), 1.0.times.10.sup.-5 M pepstatin A (isovaleryl-Val-Val-4-amino-3-hydroxy-6-methyl-heptanoyl-Ala-4-amino-3-hydroxy-6-methylheptanoic acid, ICN Biomedicals, CA), 1.0.times.10.sup.-5 M chymostatin (Boehringer), 1.0.times.10.sup.-5 M leupeptin (ICN), 5.times.10.sup.-5 M AEBSF (4-(2-aminoethyl)-benzenesulfonyl fluoride, ICN), and 5% (v/v) glycerol. Approximately 4 ml of homogenization buffer was used to homogenize each gram of frozen worms (approximately 500 worms). Insoluble material was pelleted by two sequential centrifugation steps: 19,000.times.g.sub.max at 4.degree. C. for 30 minutes followed by 110,000.times.g.sub.max at 4.degree. C. for 40 minutes. The supernatant solution was clarified by passage through a 0.45 micrometer cellulose acetate filter (Corning, N.Y.) to give Ancylostoma caniumum lysate.
(B) Concanavalin A Sepharose Chromatography.
Ancylostoma caniumum lysate (100 ml) was adsorbed onto 22 ml of Concanavalin A Sepharose (Pharmacia, Sweden) pre-equilibrated with Con A buffer (0.02M TrisHCl, pH 7.4, 1M NaCl, 0.002M CaCl.sub.2) by loading it onto a 1.6.times.11 cm column of this gel at a flow rate of 3 ml/minute (90 cm/hour). The column was at ambient temperature while the reservoir of lysate was maintained at ice bath temperature throughout the procedure. The column was subsequently washed with 2 column volumes of Con A buffer. The column flow-through and wash were collected (approximately 150 ml) and stored at -80.degree. C. until further processing was done.
(C) Anion-Exchange Chromatography.
The flow-through and wash of the Concanavalin A Sepharose column was buffered by adding solid sodium acetate to a final concentration of 12.5 mM. The conductivity was reduced by dilution with milliQ water and the pH was adjusted with HCl to pH 5.3. The precipitate formed during pH adjustment was pelleted by centrifugation 15,000.times.g.sub.max at 4.degree. C. for 15 minutes. The supernatant solution was clarified by passage through a 0.2 micrometer cellulose acetate filter (Corning, N.Y.).
This clarified solution (total volume approximately 600 ml) was loaded on to a Poros20 HQ (Perseptive Biosystems, Mass.) 1.times.2 cm column pre-equilibrated with Anion buffer (0.05M Na acetate, pH 5.3, 0.1M NaCl) at a flow rate of 10 ml/minute (800 cm/hour). The column and the solution added were at ambient temperature throughout this purification step. The column was subsequently washed with 10 column volumes of Anion buffer. Material that had inhibitory activity in the factor Xa amidolytic assay was eluted with Cation buffer containing 0.55M NaCl at a flow rate of 5 ml/minute (400 cm/hour).
(D) Heat Treatment.
Half of the 0.55M NaCl elution pool (3 ml) from anion-exchange chromatography was neutralized by adding 1M Tris-HCl, pH 7.5 to a final concentration of 50 mM, incubated for 5 minutes at 90.degree. C. in a glass tube and subsequently cooled rapidly on ice. Insoluble material was pelleted by centrifugation 19,000.times.g.sub.max at 4.degree. C. for 20 minutes. The supernatant contained material which inhibited factor Xa in the factor Xa amidolytic assay. About 89% of the factor Xa inhibitory activity was recovered in the supernatant, after this heat treatment after accounting for dilution.
(E) Molecular Sieve Chromatography using Superdex30 (alternative for the heat treatment step).
Half of the 0.55M NaCl elution pool (3 ml) from anion-exchange chromatography was loaded on a Superdex30 PG (Pharmacia, Sweden) 1.6.times.66 cm column pre-equilibrated with 0.01M sodium phosphate, pH 7.4, 0.15M NaCl at 24.degree. C. The chromatography was conducted at a flow rate of 2 ml/minute. The factor Xa inhibitory activity (determined in the factor Xa amidolytic assay) eluted 56-64 ml into the run (K.sub.av Of 0.207). This elution volume would be expected for a globular protein with a molecular mass of 14,000 daltons.
(F) Reverse Phase Chromatography.
Hookworm lysate which was fractionated by chromatography on Concanavalin A Sepharose, anion-exchange and Superdex30 (or with the alternative heat treatment step) was loaded on to a 0.46.times.25 cm C18 column (218TP54 Vydac) which was then developed with a linear gradient of 10-35% acetonitrile in 0.1% (v/v) trifluoroacetic acid at a flow rate of 1 ml/minute with a rate of 0.625% change in acetonitrile/minute. FXa inhibitory activity (determined in the factor Xa amidolytic assay) eluted at approximately 30% acetonitrile. The HPLC runs were performed on a Vista 5500 connected with a Polychrom 9600 detector set at 215 nm (Varian, Calif.). Detector signals were integrated on a 4290 integrator obtained from the same company. Factor Xa inhibitory activity containing fractions were vacuum dried and then redissolved in PBS (0.01M sodium phosphate, pH 7.4, 0.15M NaCl).
These fractions were pooled and then loaded on to a 0.46.times.25 cm C18 column (218TP54 Vydac) which was developed with a linear gradient of 10-35% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/minute with a slower rate of 0.4% change in acetonitrile/minute. Factor Xa inhibitory activity containing fractions were pooled and subsequently vacuum dried.
(G) Molecular Weight Determination of NAP from Ancylostoma caninum.
The estimated mass for NAP isolated as described in this example was determined using electrospray ionisation mass spectrometry.
A vacuum-dried pellet of NAP was dissolved in 50% (v/v) acetonitrile, 1% (v/v) formic acid. Mass analysis was performed using a VG Bio-Q (Fisons Instruments, Manchester UK).
The NAP sample was pumped through a capillary and at its tip a high voltage of 4 kV was applied. Under the influence of the high electric field, the sample was sprayed out in droplets containing the protein molecules. Aided by the drying effect of a neutral gas (N.sub.2) at 60.degree. C., the droplets were further reduced in size until all the solvent had been evaporated and only the protein species remained in the gaseous form. A population of protein species arose which differed from each other in one charge. With a quadrupole analyzer, the different Da/e (mass/charge)-values were detected. Calibration of the instrument was accomplished using Horse Heart Myoglobin (Sigma, Mo.).
The estimated mass of NAP isolated as described in sections A, B, C, D, and F of this example is 8734.60 daltons. The estimated mass of native NAP isolated as described in sections A, B, C, E, and F is 8735.67 daltons.
(H) Amino Acid Sequencing of NAP from Ancylostoma caninum.
Amino acid determination was performed on a 476-A Protein/Peptide Sequencer with On Board Microgradient PTH Analyzer and Model 610A Data Analysis System (Applied Biosystems, CA). Quantification of the residues was performed by on-line analysis on the system computer (Applied Biosystems, CA); residue assignment was performed by visual analysis of the HPLC chromatograms. The first twenty amino acids of the amino-terminus of native NAP were determined to be:
Lys Ala Tyr Pro Glu Cys Gly Glu Asn Glu Trp Leu Asp Asp Cys Gly Thr Gln Lys Pro �SEQ. ID. NO. 10!.
The cysteine residues were not directly detected in this analysis because the sample was not reduced and subsequently alkylated. Cysteines were assigned to the positions where no specific amino acid was identified.
Example 2
Cloning and Sequencing of NAP from Ancylostoma caninum.
(A) Preparation Of Hybridization Probe.
Full-length cDNA clones encoding NAP were isolated by screening a cDNA library, prepared from the mRNA isolated from the nematode, the dog hookworm, Ancylostoma caninum, using as a hybridization probe--a radiolabeled degenerate oligonucleotide whose sequence was based on the first eleven amino acids of the amino-terminus of NAP from A. caninum:
Lys Ala Tyr Pro Glu Cys Gly Glu Asn Glu Trp �SEQ. ID. NO. 7!.
The 33-mer oligonucleotide hybridization probe, designated YG99, had the following sequence:
AAR GCi TAY CCi GAR TGY GGi GAR AAY GAR TGG �SEQ. ID. NO. 1!
where "R" refers to A or G; "Y" refers to T or C; and "i" refers to inosine. YG99 was radiolabeled by enzymatic 5'-end phosphorylation (5'-end labeling kit; Ainersham, Buckinghamshire, England) using gamma-.sup.32 P-ATP (specific activity >7000 Ci/mmole; ICN, Costa Mesa, Calif., USA) and subsequently passed over a NAP.TM.10 column (Pharmacia, Uppsala, Sweden).
(B) Preparation of cDNA Library.
A cDNA library was constructed using described procedures (Promega Protocols and Applications Guide 2nd Ed.; Promega Corp., Madison, Wis., USA).
Adult canine hookworms, Ancylostoma caninum, were obtained from Yale University, Dr. Peter Hotez. Poly(A+) RNA was prepared using the QuickPrep mRNA Purification Kit (Pharmacia). About 3 micrograms of mRNA was reverse transcribed using an oligo(dT)-NotI primer/adaptor, AATTCGCGGCCGC(T).sub.15 �SEQ. ID. NO. 8!, (Promega Corp.) and AMV (Avian Myeloblastosis Virus) reverse transcriptase (Boehringer, Mannheim, Germany). The enzymes used for double-stranded cDNA synthesis were the following: E. coli DNA polymerase I and RNaseH from Life Technologies (Gaithersburg, Md., USA) and T4 DNA polymerase from Pharmacia.
EcoRI linkers (pCGGAATTCCG) �SEQ. ID. NO. 11! were ligated onto the obtained cDNA after treatment with EcoRI methylase (RiboClone EcoRI Linker Ligation System; Promega).
The cDNAs were digested with NotI and EcoRI, passed over a 1.5% agarose gel (all sizeable material was eluted using the Geneclean protocol, BIO101 Inc., La Jolla, Calif.), and unidirectionally ligated into the EcoRI-NotI arms of the lambda gt11 Sfi-NotI vector (Promega). After in vitro packaging (GigapackII-Gold, Stratagene, La Jolla, Calif.) recombinant phage were obtained by infecting strain Y1090 (Promega).
The usefulness of the cDNA library was demonstrated by PCR analysis (Taq polymerase from Boehringer; 30 temperature cycles: 1 minute at 95.degree. C.; 1 minute at 50.degree. C.; 3 minutes at 72.degree. C.) of a number of randomly picked clones using the lambda gt11 primer #1218, having the sequence, GGTGGCGACG ACTCCTGGAG CCCG �SEQ. ID. NO. 9! (New England Biolabs, Beverly, Mass., USA; targeting sequences located upstream of the cDNA insert) in combination with the above-mentioned oligo(dT)-NotI primer/adaptor; the majority of the clones was found to contain cDNA inserts of variable size.
(C) Identification of Clones.
Approximately 1.times.10.sup.6 cDNA clones (duplicate plaque-lift filters were prepared using Hybond.TM.-N; Amersham) were screened with the radiolabeled YG99 oligonucleotide using the following pre-hybridization and hybridization conditions: 5.times. SSC (SSC: 150 mM NaCl, 15 mM trisodium citrate), 5.times. Denhardt's solution, 0.5% SDS, 100 micrograms/ml sonicated fish sperm DNA (Boehringer), overnight at 42.degree. C. The filters were washed 4 times in 2.times. SSC, 0.1% SDS at 37.degree. C. After exposure (about 72 hours) to X-ray film, a total of between 350 and 500 hybridization spots were identified.
Twenty-four positive clones, designated NAP1 through NAP24, were subjected to a second hybridization round at lower plaque-density; except for NAP24, pure isolated clones were identified. The retained clones were analyzed by PCR amplifications (Taq polymerase from Boehringer; 30 temperature cycles: 1 minute at 95.degree. C.; 1 minute at 50.degree. C.; 1.5 minutes at 72.degree. C.) using the oligo(dT)-NotI primer (AATTCGCGGC CGC(T).sub.15 �SEQ. ID. NO. 8!) in combination with either (i) YG99 or (ii) the lambda gt11 primer #1218, having the sequence, GGTGGCGACG ACTCCTGGAG CCCG �SEQ. ID. NO. 9! (New England Biolabs). The majority of the clones (20 out of 23) yielded a fragment of about 400 bp when the oligo(dT)-NotI/YG99 primer set was used and a fragment of about 520 bp when the oligo(dT)-NotI/#1218 primer couple was used. Nineteen such possibly full-length clones were further characterized.
The cDNA inserts of five clones were subcloned as SfiI-NoI fragments on both pGEM-5Zf(-) and pGEM-9Zf(-) (Promega). Because the SfiI sites of lambda gt11 and pGEM-5Zf(-) are not compatible with one another, the cloning on this vector required the use of a small adaptor fragment obtained after annealing the following two 5'-end phosphorylated oligonucleotides: pTGGCCTAGCG TCAGGAGT �SEQ. ID. NO. 12! and pCCTGACGCTA GGCCATGG �SEQ. ID. NO. 13!. Following preparation of single-stranded DNA, the sequence of these cDNAs was determined with the dideoxy chain termination method using primer #1233 having the sequence, AGCGGATAAC AATTTCACAC AGGA �SEQ. ID. NO. 14! (New England Biolabs). All five clones were found to be full-length including a complete secretion signal. Clones NAP5, NAP7 and NAP22 were found to have an identical coding region. Clones NAP6 and NAP11 are also identical but differ from the NAP5 type of coding region. FIG. 1 depicts the nucleotide sequence of the NAP5 gene and FIG. 2 depicts the amino acid sequence of the protein encoded, NAP isoform 5. Likewise, FIG. 3 depicts the nucleotide sequence of the NAP6 gene and FIG. 4 depicts the amino acid sequence of the protein encoded, NAP isoform 6.
Fourteen other possibly full-length clones were subjected to a restriction analysis. The above mentioned 400 bp PCR product obtained with the YG99/oligo(dT)-NotI primer couple, was digested with four different enzymes capable of discriminating between a NAP5- and NAP6-type of clone: Sau96I, Sau3AI, DdeI, and HpaIII. The results were consistent with 10 out of the 14 clones being NAP5-type (e.g. NAP4, NAP8, NAP9, NAP15, NAP16, NAP17, NAP18, NAP20, NAP21, and NAP23) while the remaining four were NAP6-type (e.g. NAP10, NAP12, NAP14, and NAP19).
Example 3
Production and Purification Of Recombinant NAP Isoform 5 In P. pastoris.
(A) Expression Vector Construction.
The Pichia pastoris yeast expression system, including the E. coli/P. pastoris shuttle vector, pHILD2, has been described in a number of United States Patents. See, e.g., U.S. Pat. Nos. 5,330,901; 5,268,273; 5,204,261; 5,166,329; 5,135,868; 5,122,465; 5,032,516; 5,004,688; 5,002,876; 4,895,800; 4,885,242; 4,882,279; 4,879,231; 4,857,467; 4,855,231; 4,837,148; 4,818,700; 4,812,405; 4,808,537; 4,777,242; and 4,683,293.
The pYAM7SP8 vector used to direct expression and secretion of recombinant NAP in P. pastoris was a derivative of the pHILD2 plasmid (Despreaux, C. W. and Manning, R. F., Gene 131: 35-41 (1993)), having the same general structure. In addition to the transcription and recombination elements of pHILD2 required for expression and chromosomal integration in P. pastoris (see Stroman, D. W. et al., U.S. Pat. No. 4,855,231), this vector contained a chimeric prepro leader sequence inserted downstream of the alcohol oxidase (A0X1) promoter. The prepro leader consisted of the acid phosphatase (PHO1) P. pastoris secretion signal fused to a synthetic 19-amino acid pro-sequence. This pro-sequence was one of the two 19-aa pro-sequences designed by Clements et al., Gene 106: 267-272 (1991) on the basis of the Saccharomyces cerevisiae alpha-factor leader sequence. Engineered immediately downstream from the prepro leader sequence was a synthetic multi-cloning site with recognition sequences for the enzymes StuI, SacII, EcoRI, BglII, NotI, XhoI, SpeI and SpeI to facilitate the cloning of foreign genes. NAP as expressed from pYAM7SP8 in Pichia pastoris was first translated as a prepro-product and subsequently processed by the host cell to remove the pre- and pro- sequences.
The structure of this vector is shown in FIG. 12. The signal sequence (S) has the nucleic acid sequence: ATG TTC TCT CCA ATT TTG TCC TTG GAA ATT ATT TTA GCT TTG GCT ACT TTG CAA TCT GTC TTC GCT �SEQ. ID. NO. 15!. The pro sequence (P) has the nucleic acid sequence: CAG CCA GGT ATC TCC ACT ACC GTT GGT TCC GCT GCC GAG GGT TCT TTG GAC AAG AGG �SEQ. ID. NO. 16!. The multiple cloning site (MCS) has the nucleic acid sequence: CCT ATC CGC GGA ATT CAG ATC TGA ATG CGG CCG CTC GAG ACT AGT GGA TCC �SEQ. ID. NO. 17!.
The pGEM-9Zf(-) vector (Promega) containing the NAP5 cDNA was used to isolate by amplification ("PCR-rescue") the region encoding the mature NAP isoform 5 protein (using Vent polymerase from New England Biolabs, Beverly, Mass.; 20 temperature cycles: 1 minute at 94.degree. C., 1 minute at 50.degree. C., and 1.5 minutes at 72.degree. C.). The following oligonucleotide primers were used:
YG101 �SEQ. ID. NO. 18!: GCTCGCTCTA-GAAGCTTCAG-ACATGTATAA-TCTCATGTTG-G
YG103 �SEQ. ID. NO. 19!: AAGGCATACC-CGGAGTGTGG-TG The YG101 primer, targeting C-terminal sequences, contained a non-annealing extension which included XbaI and HindIII restriction sites (underlined).
Following digestion with XbaI enzyme, the amplification product, having the expected size, was isolated from gel and subsequently enzymatically phosphorylated (T4 polynucleotide kinase from New England Biolabs, Beverly, Mass.). After heat-inactivation (10 minutes at 70.degree. C.) of the kinase, the blunt-ended/XbaI fragment was directionally cloned into the vector pYAM7SP8 for expression purposes. The recipient vector-fragment from pYAM7SP8 was prepared by StuI-SpeI restriction, and purified from agarose gel. The E. coli strain, WK6 �Zell, R. and Fritz, H.-J., EMBO J., 6: 1809-1815 (1987)!, was transformed with the ligation mixture, and ampicillin resistant clones were selected.
Based on restriction analysis, a plasmid clone containing an insert of the expected size, designated pYAM7SP-NAP5, was retained for further characterization. Sequence determination of the clone pYAM7SP-NAP5 confirmed the precise insertion of the mature NAP5 coding region in fusion with the prepro leader signal, as predicted by the construction scheme, as well as the absence of unwanted mutations in the coding region.
(B) Expression Of Recombinant NAP Isoform 5 In P. pastoris.
The Pichia strain GTS115 (his4) has been described in Stroman, D. W. et al., U.S. Pat. No. 4,855,231. All of the P. pastoris manipulations were performed essentially as described in Stroman, D. W. et al., U.S. Pat. No. 4,855,231.
About 1 microgram of pYAM7SP-NAP5 plasmid DNA was electroporated into the strain GTS115 using a standard electroporation protocol. The plasmid was previously linearized by SalI digestion, theoretically targeting the integration event into the his4 chromosomal locus.
The selection of a NAP isoform 5 high-expresser strain was performed essentially as described hereinbelow. His+ transformants were recovered on MD plates (Yeast Nitrogen Base without amino acids (DIFCO), 13.4 g/l; Biotin, 400 micrograms/L; D-glucose, 20 g/l; agar, 15 g/1). Single colonies (n=60) originating from the electroporation were inoculated into 100 microliters of FM22-glycerol-PTM1 medium in wells of a 96-well plate and were allowed to grow on a plate-agitator at 30.degree. C. for 24 hours. One liter of FM22-glycerol-PTM1 medium contained 42.87 g KH.sub.2 PO.sub.4, 5 g (NH.sub.4).sub.2 SO.sub.4, 1 g CaSO.sub.4.2H.sub.2 O, 14.28 g K.sub.2 SO.sub.4, 11.7 g MgSO.sub.4.7H.sub.2 O, 50 g glycerol sterilized as a 100 ml solution, and 1 ml of PTM1 trace mineral mix filter-sterilized. The FM22 part of the medium was prepared as a 900 ml solution adjusted to pH 4.9 with KOH and sterile filtered. One liter of the PTM1 mix contained 6 g CuSO.sub.4.5H.sub.2 O, 0.8 g KI, 3 g MnSO.sub.4.H.sub.2 O, 0.2 g NaMoO.sub.4.2H.sub.2 O, 0.02 g H.sub.3 BO.sub.3, 0.5 g COCl.sub.2.6H.sub.2 O, 20 g ZnCl.sub.2, 5 ml H.sub.2 SO.sub.4, 65 g FeSO.sub.4.7H.sub.2 O, 0.2 g biotin.
The cells were then pelleted and resuspended in fresh FM22-methanol-PTM1 medium (same composition as above except that the 50 g glycerol was replaced by 0.5% (v/v) methanol in order to induce the AOX1 promoter). After an additional incubation period of 24 hours at 30.degree. C., the supernatants of the mini-cultures were tested for the presence of secreted NAP isoform 5. Two clones were selected that directed high synthesis and secretion of NAP isoform 5, as was shown by the appearance of high factor Xa inhibitory activity in the culture medium (as measured by the amidolytic factor Xa assay). After a second screening round, using the same procedure, but this time at the shake-flask level, one isolated host cell was chosen and designated P. pastoris GTS115/7SP-NAP5.
The host cell, GTS115/7SP-NAP5, was shown to have a wild type methanol-utilisation phenotype (Mut.sup.+), which demonstrated that the integration of the expression cassette into the chromosome of GTS115 did not alter the functionality of the genomic AOX1 gene.
Subsequent production of recombinant NAP isoform 5 material was performed in shake flask cultures, as described in Stroman, D. W. et al., U.S. Pat. No. 4,855,231. The recombinant product was purified from Pichia pastoris cell supernatant as described below.
(C) Purification of NAP Isoform 5.
(1) Cation Exchange Chromatography.
The culture supernatant (100 ml) was centrifuged at 16000 r.p.m. (about 30,000.times.g) for 20 minutes before the pH was adjusted with 1N HCl to pH 3. The conductivity of the supernatant was decreased to less than 10 mS/cm by adding MilliQ water. The diluted supernatant was clarified by passage through a 0.22 micrometer cellulose acetate filter (Corning Inc., Corning, N.Y., USA)
The total volume (approximately 500 ml) of supernatant was loaded on a Poros20 HS (Perseptive Biosystems,MA) 1.times.2 cm column pre-equilibrated with Cation Buffer (0.05M sodium citrate, pH 3) at a flow rate of 5 ml/minute (400 cm/hour). The column and the sample were at ambient temperature throughout this purification step. The column was subsequently washed with 50 column volumes Cation Buffer. Material that had inhibitory activity in a factor Xa amidolytic assay was eluted with Cation Buffer containing 1M NaCl at a flow rate of 2 ml/minute.
(2) Molecular Sieve Chromatography Using Superdex30.
The 1M NaCl elution pool containing the inhibitory material (3 ml) from the cation-exchange column was loaded on a Superdex30 PG (Pharmacia, Sweden) 1.6.times.66 cm column pre-equilibrated with 0.01M sodium phosphate, pH 7.4, 0.15M NaCl at ambient temperature. The chromatography was conducted at a flow rate of 2 ml/minute. The factor Xa inhibitory activity eluted 56-64 ml into the run (K.sub.av of 0.207). This is exact the same elution volume as determined for the native molecule.
(3) Reverse Phase Chromatography.
1 ml of the pooled fractions from the gel filtration chromatography was loaded on to a 0.46.times.25 cm C18 column (218TP54 Vydac) which was then developed with a linear gradient of 10-35%. acetonitrile in 0.1% (v/v) trifluoroacetic acid at 1 ml/minute with a rate of 0.4% change in acetonitrile/minute. Factor Xa inhibitory activity elutes around 30-35% acetonitrile and was present in several fractions. HPLC runs were performed on the same system as described in Example 1. Fractions from several runs on this column containing the factor Xa inhibitory activity were pooled and vacuum dried.
(4) Molecular Weight Determination of NAP Isoform 5.
The estimated mass for the main constituent isolated as described in sections (1) to (3) of this example were determined using the same electrospray ionisation mass spectrometry system as described in Example 1.
The estimated mass of recombinant NAP isoform 5 is 8735.69 daltons.
(5) Amino Acid Secruencing of NAP Isoform 5.
Following purification by section (1) to (3) of this example, the recombinant NAP from Pichia pastoris was subjected to amino acid sequence analysis as described in Example 1. The first five amino acids of the aminoterminus of NAP isoform 5 were determined to be: Lys-Ala-Tyr-Pro-Glu �SEQ. ID. NO. 20!. Compared to the authentic NAP protein (see Example 1), the sequence were found to be identical.
Example 4
Production and Purification Of Recombinant NAP Isoform 6 In P. pastoris.
(A) Expression Vector Construction.
The expression vector, pYAM7SP-NAP6, was made in the same manner as described in Example 3.
(B) Expression Of Recombinant NAP Isoform 6 In P. pastoris.
The vector, pYAM7SP-NAP6, was used to transform the Pichia strain GTS115 (his4) as described in Example 3.
(C) Purification of NAP Isoform 6.
The recombinant NAP isoform 6, expressed from Pichia strain GTS115 (his4) transformed with the expression vector, pYAM7SP-NAP6, was purified as described in Example 3.
The estimated mass of recombinant NAP isoform 6 was determined to be 8393.84 daltons.
The majority of the NAP isoform 6 preparation had the following amino-terminus: Lys-Ala-Tyr-Pro-Glu �SEQ. ID. NO. 20!.
Example 5
Expression Of Recombinant Pro-NAP Isoform 5 In COS Cells
(A) Expression Vector Construction.
The pGEM-9Zf(-) vector (Promega Corporation, Madison, Wis., USA) onto which the NAP5 cDNA was subcloned, served as target for PCR-rescue of the entire NAP isoform 5 coding region, including the native secretion signal (using Vent polymerase from New England Biolabs, Beverly, Mass., USA; 20 temperature cycles: 1 minute at 95.degree. C., 1 minute at 50.degree. C., and 1.5 minutes at 72.degree. C.) The oligonucleotide primers used were: (1) YG101, targeting the 3'-end of the gene encoding a NAP and having the sequence, GCTCGCTCTA GAAGCTTCAG ACATGTATAA TCTCATGTTG G �SEQ. ID. NO. 18!, and (2) YG102, targeting the 5'-end of the gene encoding a NAP and having the sequence, GACCAGTCTA GACAATGAAG ATGCTTTACG CTATCG �SEQ. ID. NO. 21!. These primers contain non-annealing extensions which include XbaI restriction sites (underlined).
Following digestion with XbaI enzyme, the amplification product having the expected size was isolated from an agarose gel and subsequently substituted for the about 450 basepair XbaI stuffer fragment of the pEF-BOS vector �Mizushima, S. and Nagata, S., Nucl. Acids Res., 18: 5322 (1990)! for expression purposes. The recipient vector-fragment was prepared by XbaI digestion and purified from an agarose gel.
E. coli strain WK6 �Zell, R. and Fritz, H.-J., EMBO J., 6: 1809-1815 (1987)! was transformed with the ligation mixture. Thirty randomly picked ampicillin-resistant transformants were subjected to PCR analysis (Taq polymerase from Life Technologies Inc., Gaithersburg, Md., USA; 30 cycles of amplification with the following temperature program: 1 minute at 95.degree. C., 1 minute at 50.degree. C., and 1 minute at 72.degree. C.). Primers used were: (i) YG103 having the sequence, AAGGCATACC CGGAGTGTGG TG �SEQ. ID. NO. 19!, and matching the amino-terminus of the region encoding mature NAP, and (ii) YG60 having the sequence, GTGGGAGACC TGATACTCTC AAG �SEQ. ID. NO. 22!, and targeting vector sequences downstream of the site of insertion, i.e., in the 3'-untranslated region of the pEF-BOS expression cassette. Only clones that harbor the insert in the desired orientation can yield a PCR fragment of predictable length (about 250 basepair). Two such clones were further characterized by sequence determination and were found to contain the intended XbaI insert. One of the clones, designated pEF-BOS-NAP5, was used to transfect COS cells.
(B) Transfection of COS Cells.
COS-7 cells (ATCC CRL 1651) were transfected with pEF-BOS-NAP5, PEF-BOS containing an irrelevant insert or with omission of DNA (mock transfections) using DEAE-dextran. The following media and stock solutions are used with the DEAE-dextran method:
(1) COS-medium: DMEM; 10% FBS (incubated for 30 minutes at 56.degree. C.); 0.03% L-glutamine; penicillin (50 I.U./ml) and streptomycin (50 micrograms/ml) (all products from Life Technologies).
(2) MEM-HEPES: MEM medium from Life Technologies Inc., reconstituted according to the manufacturer's specifications; containing a 25 mM final concentration of HEPES; adjusted to pH 7.1 before filtration (0.22 micrometer).
(3) DNA solution: 6 micrograms DNA per 3 ml MEM-HEPES
(4) DEAE-dextran solution: 30 microliters DEAE-dextran stock (Pharmacia, Uppsala, Sweden; 100 mg/ml in H.sub.2 O) per 3 ml MEM-HEPES.
(5) Transfection mixture: 3 ml of the DEAE-dextran solution is added to 3 ml of the DNA solution and the mixture is left to stand for 30 minutes at ambient temperature.
(6) Chloroquine solution: a 1:100 dilution of chloroquine stock (Sigma, St.Louis, Mo., USA; 10 mM in water; filtered through a 0.22 micrometer membrane) in COS medium.
Transient transfection of the COS cells is performed as follows. COS cells (about 3.5.times.10.sup.6), cultured in a 175 cm.sup.2 Nunc TC-flask (Life Technologies Inc.) were washed once with MEM-HEPES. Six ml of the Transfection mixture was pipetted onto the washed cells. After incubation for 30 minutes at ambient temperature, 48 ml of the chloroquine solution was added and the cells were incubated for another 4 hours at 37.degree. C. The cells were washed one time with fresh COS-medium and finally incubated in 50 ml of the same medium at 37.degree. C.
(C) Culturing of Transfected COS Cells.
Three, four, and five days after transfection a sample of the culture supernatants was tested in a factor Xa amidolytic assay. Reaction mixtures (150 microliters) were prepared in 96-well plates containing factor Xa and various dilutions of the culture supernatants in assay buffer (100 mM Tris-HCl pH 7.4; 140 mM NaCl; 0.1% BSA). Human factor X was purchased from Enzyme Research Laboratories (South Bend, Ind., USA) and activated with Russell's Viper venom using the procedure of Bock, P. E., Craig, P. A., Olson, S. T., and Singh P., Arch. Biochem. Biophys., 273: 375-388 (1989). Following a 30 minute incubation at ambient temperature, the enzymatic reactions were initiated by addition of 50 microliters of a 1 mM substrate solution in water (N-alpha-benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine p-nitroanilidedihydrochloride; S-2765; Chromogenix, Molndal, Sweden) to yield final concentrations of 0.2 nM factor Xa and 0.25 mM S-2765. Substrate hydrolysis was monitored by continuously measuring absorbance at 405 nm using a Vmax kinetic plate reader (Molecular Devices, Menlo Park, Calif., USA).
The results clearly demonstrated that factor Xa inhibitory activity was accumulating in the culture supernatant of the cells transfected with pEF-BOS-NAP5. From the inhibitory activity measurements, it was estimated that the NAP in the culture medium amounts to about 200 micrograms per liter.
The COS culture supernatant was harvested five days after transfection and the NAP protein purified as described in Example 6.
Example 6
Purification Of Recombinant Pro-NAP Isoform 5.
(A) Anion Exchange Chromatography.
The COS culture supernatant containing Pro-NAP was centrifuged at 1500 r.p.m. (about 500.times.g) for 10 minutes before adding solid sodium acetate to a final concentration of 50 mM. The following protease inhibitors were added (all protease inhibitors from ICN Biomedicals Inc, Costa Mesa, Calif., USA): 1.0.times.10.sup.-5 M pepstatin A (isovaleryl-Val-Val-4-amino-3-hydroxy-6-methyl-heptanoyl-Ala-4-amino-3-hydroxy-6-methylheptanoic acid), 1.0.times.10.sup.-5 M leupeptin, 5.times.10.sup.-5 M AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride). The pH was adjusted with HCl to pH 5.3. The supernatant was clarified by passage through a 0.2 micrometer cellulose acetate filter (Corning Inc., Corning, N.Y., USA).
The clarified supernatant (total volume approximately 300 ml) was loaded on a Poros20 HQ (Perseptive Biosystems, MA) 1.times.2 cm column pre-equilibrated with Anion buffer (0.05M sodium acetate, pH 5.3, 0.1M NaCl) at a flow rate of 10 ml/minute (800 cm/hour). The column and the sample were at ambient temperature throughout this purification step. The column was subsequently washed with at least 10 column volumes of Anion buffer. Material that had inhibitory activity in a factor Xa amidolytic assay was eluted with Anion buffer containing 0.55M NaCl at a flow rate of 5 ml/minute (400 cm/hour) and was collected.
(B) Molecular Sieve Chromatography Using Superdex30.
The 0.55M NaCl elution pool (3 ml) from the anion-exchange chromatography was loaded on a Superdex30 PG (Pharmacia, Sweden) 1.6.times.66 cm column pre-equilibrated with 0.01M sodium phosphate, pH 7.4, 0.15M NaCl at 24.degree. C. The chromatography was conducted at a flow rate of 2 ml/minute. Material which was inhibitory in the Factor Xa amidolytic assay eluted 56-64 ml into the run (K.sub.av of 0.207). This was exactly the same elution volume as determined for the native molecule.
(C) Heat Treatment.
The total pool of fractions having factor Xa inhibitory activity was incubated for 5 minutes at 90.degree. C. in a glass tube and subsequently cooled rapidly on ice. Insoluble material was pelleted by centrifugation 19,000.times.g.sub.max at 4.degree. C. for 20 minutes. The supernatant contained all of the factor Xa inhibitory activity.
(D) Reverse Phase HPLC Chromatography.
The supernatant of the heat-treated sample was loaded onto a 0.46.times.25 cm C18 column (218TP54 Vydac) which was then developed with a linear gradient of 10-35%. acetonitrile in 0.1% (v/v) trifluoroacetic acid at 1 ml/minute with a rate of 0.4% change in acetonitrile/minute. Factor Xa inhibitory activity eluted at approximately 30%. acetonitrile. The HPLC runs were performed on the same system as described in Example 1. Factor Xa inhibitory activity containing-fractions were vacuum dried.
(E) Molecular Weight Determination.
The estimated mass for recombinant Pro-NAP, isolated as described in sections A-D of this example, was determined using the same electrospray ionisation mass spectrometry system as described in Example 1.
The estimated mass of recombinant Pro-NAP isoform 5 was 9248.4 daltons.
(F) Amino Acid Sequencing.
Following purification, the recombinant Pro-NAP from COS cells was subjected to amino acid analysis to determine its amino-terminus sequence, as described in Example 1. The first nine amino acids of the amino-terminus of Pro-NAP was determined to be: Arg Thr Val Arg Lys Ala Tyr Pro Glu �SEQ. ID. NO. 23!. Compared to the native NAP protein (see Example 1), Pro-NAP possesses a four additional amino acids on its N-terminus. The amino acid sequence of Pro-NAP isoform 5 is shown in FIG. 5.
Example 7
Expression Of Recombinant Pro-NAP Isoform 6 In COS Cells
Pro-NAP isoform 6 was transiently produced in Cos cells essentially as described for Pro-NAP isoform 5 in Example 5.
The NAP6 coding region, including the secretion signal, was PCR-rescued with the same two oligonucleotide primers used for NAP5: (1) YG101 �SEQ. ID. NO. 18! targeting the 3'-end of the gene and having the sequence, GCTCGCTCTA GAAGCTTCAG ACATGTATAA TCTCATGTTG G, and (2) YG102 �SEQ. ID. NO. 22! targeting the 5'-end of the gene and having the sequence, GACCAGTCTA GACAATGAAG ATGCTTTACG CTATCG. The YG101-primer contains a non-matching nucleotide when used with NAP6 as target (underlined T-residue; compare with FIG. 1 and FIG. 3); this mismatch results in the replacement an ATT Ile-codon by a ATA Ile-codon. The mismatch did not markedly influence the amplification efficiency.
The following modification was introduced: twenty-four hours after transfection of the COS cells (which is described in Example 5, section B) the COS-medium containing 10% FBS was replaced 50 ml of a medium consisting of a 1:1 mixture of DMEM and Nutrient Mixture Ham's F-12 (Life Technologies). The cells were then further incubated at 37.degree. C. and the production of factor Xa inhibitory activity detected as described in Example 5.
Example 8
Purification Of Recombinant Pro-NAP Isoform 6.
(A) Anion Exchange Chromatography.
The COS culture supernatant containing Pro-NAP was centrifuged at 1500 r.p.m. for 10 minutes before adding solid sodium acetate to a final concentration of 50 mM. The following protease inhibitors were added (all protease inhibitors from ICN Biomedicals Inc, Costa Mesa, Calif., USA): 1.0.times.10.sup.-5 M pepstatin A (isovaleryl-Val-Val-4-amino-3-hydroxy-6-methyl-heptanoyl-Ala-4-amino-3-hydroxy-6-methylheptanoic acid), 1.0.times.10.sup.-5 M leupeptin, 5.times.10.sup.-5 M AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride). The pH was adjusted with HCl to pH 5.3. The supernatant was clarified by passage through a 0.2 micrometer cellulose acetate filter (Corning Inc., Corning, N.Y., USA).
The clarified supernatant (total volume approximately 450 ml) was loaded on a Poros20 HQ (Perseptive Biosystems,MA) 1.times.2 cm column pre-equilibrated with Anion buffer (0.05M Na sodium acetate, pH 5.3, 0.1M NaCl) at a flow rate of 10 ml/minute (800 cm/hour). The column and the sample were at ambient temperature throughout this purification step. The column was subsequently washed with at least 10 column volumes of Anion buffer. Material that had inhibitory activity in a factor Xa amidolytic assay was eluted with Anion buffer containing 0.55M NaCl at a flow rate of 5 ml/minute (400 cm/hour) and was collected.
(B) Molecular Sieve Chromatography Using Superdex30.
The 0.55M NaCl elution pool (3 ml) from the anion-exchange chromatography was loaded on a Superdex30 PG (Pharmacia, Sweden) 1.6.times.66 cm column pre-equilibrated with 0.01M sodium phosphate, pH 7.4, 0.15M NaCl at 24.degree. C. The chromatography was conducted at a flow rate of 2 ml/minute. Material which was inhibitory in the Factor Xa amidolytic assay eluted 56-64 ml into the run (K.sub.av of 0.207). This was exactly the same elution volume as determined for the native NAP.
(C) Reverse Phase HPLC Chromatography.
The pooled fractions from the gel filtration were loaded onto a 0.46.times.25 cm C18 column (218TP54 Vydac) which was then developed with a linear gradient of 10-35% acetonitrile in 0.1% (v/v) trifluoroacetic acid at a flow rate of 1 ml/minute with a rate of 0.4% change in acetonitrile/minute. Factor Xa inhibitory activity eluted at approximately 30% acetonitrile. The HPLC runs were performed on the same system as described in Example 1. Factor Xa inhibitory activity containing-fractions were vacuum dried.
(D) Molecular Weight Determination.
The estimated mass for recombinant Pro-NAP isoform 6, isolated as described in sections A to C of this example, was determined using the same electro spray ionisation mass spectrometry system as described in Example 1.
The estimated mass of recombinant Pro-NAP isoform 6 was 8906.9 daltons.
(E) Amino Acid Sequencing.
Following purification, the recombinant Pro-NAP isoform 6 from COS cells was subjected to amino acid sequence analysis as described in Example 1. The first five amino acids of the N-terminus of Pro-NAP isoform 6 was determined to be: Arg Thr Val Arg Lys �SEQ. ID. NO. 24!. Compared to the native NAP protein (see Example 1), Pro-NAP isoform 6 possesses a four additional amino acids on its amino-terminus. The amino acid sequence of Pro-NAP isoform 6 is shown in FIG. 6.
Example 9
The Use of NAP DNA Sequences to Isolate Genes Encoding Other NAP Proteins.
The NAP5 and NAP6 cDNA sequences (from Example 2) were used to isolate related molecules from other parasitic species by cross-hybridization.
The pGEM-9Zf(-) vectors (Promega) containing the NAP5 and NAP6 cDNAs were used to PCR-rescue the regions encoding the mature NAP proteins (Taq polymerase from Life Technologies; 20 temperature cycles: 1 minute at 95.degree. C., 1 minute at 50.degree. C., and 1.5 minutes at 72.degree. C.). The oligonucleotide primers used were: (1) YG109 �SEQ. ID. NO. 25!, targeting the C-terminal sequences of cDNA encoding NAP, and having the sequence, TCAGACATGT-ATAATCTCAT-GTTGG, and (2) YG103 �SEQ. ID. NO. 19! having the sequence, AAGGCATACC-CGGAGTGTGG-TG. The YG109 primer contains a single nucleotide mismatch (underlined T-residue; compare with the sequences shown in FIGS. 1 and 3) when used with NAP6 as target. This did not markedly influence the amplification efficiency. The correctly sized PCR products (about 230 basepairs) were both isolated from a 1.5% agarose gel. An equimolar mixture was radiolabeled by random primer extension (T7 QuickPrime kit; Pharmacia) and subsequently passed over a Bio-Spin 30 column (Bio-Rad, Richmond, Calif., USA).
Ancylostoma ceylanicum (Ace), Ancylostoma duodenale (Adu), and Heligmosomoides polygyrus (Hpo) cDNA libraries were prepared essentially as described for Ancylostoma caninum in Example 2.
Ancylostoma ceylanicum and Heligmosomoides polygyrus were obtained from Dr. D. I. Pritchard, Department of Life Science, University of Nottingham, Nottingham, UK. Ancylostoma duodenale was obtained from Dr. G. A. Schad, The School of Veterinary Medicine, Department of Pathobiology, University of Pennsylvania, Philadelphia, Pa., USA.
In each case, the cDNAs were directionally cloned as EcoRI-NotI fragments in lambda gt11. Approximately 2.times.10.sup.5 cDNA clones from each library (duplicate plaque-lift filters were prepared using Hybond.TM.-N; Amersham) were screened with the radiolabeled NAP5 and NAP6 fragments using the following prehybridization and hybridization conditions: 5.times. SSC (SSC: 150 mM NaCl, 15 mM trisodium citrate), 5.times. Denhardt's solution, 0.5% SDS, 20% formamide, 100 micrograms/ml sonicated fish sperm DNA (Boehringer), overnight at 42.degree. C. The filters were washed 4 times for 30 minutes in 2.times. SSC, 0.1% SDS at 37.degree. C. After exposure (about 60 hours) to X-ray film, a total of between 100 and 200 hybridization spots were identified in the case of Ace and Adu. A small number of very faint spots were visible in the case of the Hpo cDNA library. For each of the libraries, eight positives were subjected to a second hybridization round at lower plaque-density so as to isolate single pure clones.
The retained clones were further characterized by a PCR amplification of the cDNA-inserts using the oligo(dT)-NotI primer (Promega; this is the same primer used to prepare first strand cDNA; see Example 2) in combination with the lambda-gt11 primer #1218 having the sequence, GGTGGCGACG ACTCCTGGAG CCCG �SEQ. ID. NO. 9! (New England Biolabs; primer #1218 targets lambda sequences located upstream of the site of cDNA insertion). PCR amplifications were performed as follows: Taq polymerase from Boehringer; 30 temperature cycles: 1 minute at 95.degree. C.; 1 minute at 50.degree. C.; 1.5 minutes at 72.degree. C. Gel-electrophoretic analysis of the PCR products clearly demonstrated that cDNAs of roughly the same size as the NAP5 cDNA (e.g., 400 to 500 bp) were obtained for each species. In addition to these NAP5--sized cDNAs, some Ace and Adu cDNAs were estimated to be about 700 bp long.
A number of clones, containing either a 500 bp or an 800 bp insert, were chosen for sequence determination. To that end the cDNA inserts were subcloned, as SfiI-NotI fragments, onto pGEM-type phagemids (Promega; refer-to Example 2 for details) which permit the preparation of single stranded DNA. The sequencing results led to the identification of six different new NAP-like proteins, designated as follows: AceNAP4, AceNAP5, AceNAP7, AduNAP4, AduNAP7, and HpoNAP5. The nucleotide sequences of the cDNAs as well as the deduced amino acid sequences of the encoded proteins are shown in FIG. 7A (AceNAP4), FIG. 7B (AceNAP5), FIG. 7C (AceNAP7), FIG. 7D (AduNAP4), FIG. 7E (AduNAP7), and FIG. 7F (HpoNAP5). The AceNAP4 and AduNAP7 cDNAs, each about 700 bp long, each encoded proteins which incorporated two NAP domains; the other cDNAs isolated coded for a protein having a single NAP domain. The AduNAP4 cDNA clone was not full-length, i.e, the clone lacked the 5'-terminal part of the coding region; the correct reading frame could, however, be assigned based on amino acid sequence homology with the NAP family of related molecules.
The identified cDNA sequences can be used to produce the encoded proteins as disclosed in Examples 3, 4, 5, and 7 using the same or alternative suitable expression systems. Conditioned media or cell lysates, depending on the system used, can be tested as such or after fractionation (using such methodology as outlined in Example 3, 4, 6 and 8) for protease inhibitory and anticoagulant activity. Proteins that are encoded by cDNAs which hybridize to probes derived from fragments of the NAP5 gene (FIG. 1) and/or the NAP6 gene (FIG. 3) and that possess anticoagulant properties are considered to belong to the NAP family of related molecules.
Example 10
Identification of NAP by Functional Display of cDNA Encoded Proteins.
(A) The pDONG Series of Vectors.
The nucleotide sequences of the PDONG vectors, pDONG61, pDONG62 and pDONG63, derivatives of pUC119 �Vieira, J. and Messing, J., Methods in Enzymology, 153:311 (1987)!, are depicted in FIGS. 8A to 8C respectively.
To construct these three vectors, HindIII and SfiI restriction sites were added at the 5'-end and 3'-end of the filamentous phage gene 6 by PCR amplification of the M13KO7 single stranded DNA �Vieira, J. and Messing, J., Ibid! with the G6BACKHIND backward primer and G6FORSFI61, G6FORSFI62 or G6FORSFI63 as forward primers. In a second PCR, the three obtained fragments were re-amplified with G6BACKHIND and GGFORNOTBAMH as forward primer to append NotI and BamHI sites at the 3'-end of the fragments. The sequences of the above mentioned PCR-primers are as follows (restriction sites are underlined):
G6BACKHIND:ATCCGAAGCT TTGCTAACAT ACTGCGTAAT AAG �SEQ. ID. NO. 26!
G6FORSFI61:TATGGGATGG CCGACTTGGC CTCCGCCTGA GCCTCCACCT TTATCCCAAT CCAAATAAGA �SEQ. ID. NO. 27!
G6FORSFI62:ATGGGATGGC CGACTTGGCC CTCCGCCTGA GCCTCCACCT TTATCCCAAT CCAAATAAGA �SEQ. ID. NO. 28!
G6FORSFI63:TATGGGATGG CCGACTTGGC CGATCCGCCT GAGCCTCCAC CTTTATCCCA ATCCAAATAA �SEQ. ID. NO. 29!
GAG6FORNOTBAMH:AGGAGGGGAT CCGCGGCCGC GTGATATGGG ATGGCCGACT TGGCC �SEQ. ID. NO. 30!
Finally, the PCR products were gel-purified, individually digested with HindIII and BamHI and inserted between the corresponding sites of pUC119. Sequence determination confirmed that pDONG61, pDONG62, and pDONG63 all contained the intended insert.
The PDONG series of vectors permit the cloning of cDNAs, as SfiI-NotI fragments. This cloning fuses the cDNAs in each of the three reading frames to the 3'-end of filamentous phage gene 6 which encodes one of the phage's coat proteins. Infection of a male-specific E. coli strain harboring a pDONG-derivative, with VCSM13 helper phage (Stratagene, La Jolla, Calif.), results in the rescuing of pseudo-virions which encapsidate one specific single strand of the pDONG-derivative and which may also incorporate a recombinant protein 6 (p6) fusion protein in their coat. cDNAs which are such that the encoded protein is functionally displayed on the phage surface as a recombinant p6 fusion protein become identifiable by means of a panning experiment described below.
(B) Transfer of the Ancyloscoma caninum cDNA Library from Lambda gt11 to the pDONG Series of Vectors.
A phage lambda preparation of the pooled A. caninum cDNAclones (about 1.times.10.sup.6 plaques, see Example 2) was used to PCR-rescue the cDNA inserts (Taq polymerase from Life Technologies, Gaithersburg, Md., USA; 20 temperature cycles: 1 minute at 95.degree. C., 1 minute at 50.degree. C., and 3 minutes at 72.degree. C. followed by 10 minutes at 65.degree. C.), with the lambda gt11 primer #1218 having the sequence, GGTGGCGACG ACTCCTGGAG CCCG �SEQ. ID. NO. 9! (New England Biolabs, Beverly, Mass., USA; targeting sequences located upstream of the cDNA insert) in combination with the oligo(dT)-NotI primer/adaptor (Promega) used for first strand cDNA synthesis. Following digestion with the restriction enzymes SfiI and NotI, the whole size-range of amplification products were recovered from agarose gel.
All fragments were directionally cloned into the pDONG61, pDONG62, and pDONG63 vectors. The recipient vector-fragments were prepared by digestion of the CsCl purified vectors with SfiI and NotI and purification with the "Wizard.TM. PCR Preps DNA Purification System" (Promega Corp, Madison, Wis., USA).
E. coli strain TG1 �Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning, A Laboratory Manual, Second Edition, volumes 1 to 3, Cold Spring Harbor Laboratory Press (1989)! was transformed by electroporation with the pDONG/cDNA ligation mixtures. Electrotransformed cells were incubated 1 hour at 37.degree. C. in SOC medium (Sambrook, J. et al., Ibid.! and plated on LB-agar containing 0.1% glucose and 100 micrograms/ml carbenicillin (245.times.245.times.25 mm plates; Nunc). 2.2.times.10.sup.6, 1.6.times.10.sup.6, and 1.4.times.10.sup.6 carbenicillin resistant transformants were obtained with pDONG61, pDONG62, and pDONG63, respectively. From each library, designated 20L, 21L and 22L, a number of randomly picked transformants were subjected to PCR analysis (Taq polymerase from Life Technologies; 30 cycles of amplification with the following temperature program: 1 minute at 95.degree. C., 1 minute at 50.degree. C., and 1 to 3 minutes at 72.degree. C.) using two primers that match with sequences flanking the multiple cloning site of pUC119 (primers #1224 having the sequence, CGCCAGGGTT TTCCCAGTCA CGAC �SEQ. ID. NO. 31!, and #1233 having the sequence, AGCGGATAAC AATTTCACAC AGGA �SEQ. ID. NO. 14!; New England Biolabs). The results showed that the vast majority of the clones contained a cDNA-insert of variable size.
(C) Factor Xa Based Affinity-Selection of cDNA Clones Encoding a NAP Protein.
Phage particles from the 20L, 21L and 22L libraries were rescued as follows: each library was scraped from the plates and grown at 37.degree. C. in 100 ml LB medium supplemented with 1% glucose and 100 micrograms/ml carbenicillin until the optical absorbance at 600 nm reaches the value of 0.5. After addition of VCSM13 helper phage (Stratagene) at a multiplicity of infection (moi) of 20, the culture was left to stand for 30 minutes at 37.degree. C. and then slowly shaken for another 30 minutes. The cells were pelleted by centrifugation and resuspended in 250 ml LB medium supplemented with 100 micrograms/ml carbenicillin and 50 micrograms/ml kanamycin. These cultures were allowed to grow overnight at 30.degree. C. under vigorous agitation. The resulting phage particles were purified by two consecutive precipitations with polyethylene glycol/NaCl and resuspended at 1.times.10.sup.13 virions per ml in TRIS-buffered saline (0.05M Tris, 0.15M sodium chloride, pH 7.4) (TBS). Equal amounts of phage particles from the 20L, 21L and 22L were then mixed together.
Human factor Xa (see Example A for preparation) was biotinylated with biotin-XX-NHS (Pierce). The amidolytic activity of the protease was not affected by this modification as shown by an enzymatic assay using the chromogenic substrate S-2765 (Chromogenix; see Example 3). Streptavidin-coated magnetic beads (Dynal; 1 mg per panning round) were washed three times with TBS and blocked in TBS supplemented with 2% skim milk (Difco) at ambient temperature. After one hour, the magnetic beads were washed twice with TBS before use.
For the first round of panning, 1.times.10.sup.13 phage from the pooled libraries were incubated for 75 minutes at 4.degree. C. in 200 microliters of TBS buffer supplemented with 250 nM biotinylated factor Xa, 5 mM CaCl.sub.2 and 2% skim milk. After this time, 1 mg blocked streptavidin-coated magnetic beads, resuspended in 200 microliters of TBS containing 5 mM CaCl.sub.2 and 2% skim milk, was added to the phage solution and incubated for 1 hour at 4.degree. C. with gentle agitation. With a magnet (Dynal), the magnetic beads were then rinsed ten times with 500 microliters of TBS containing 0.1%. Tween-20. Bound phage were eluted from the magnetic beads by incubating them with 500 microliters of 0.1M glycine-HCl buffer (pH 2.0) for 10 minutes. The supernatant was neutralized with 150 microliters 1M Tris-HCl buffer (pH 8.0).
For phage propagation, E. coli strain TG1 �Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning, A Laboratory Manual, Second Edition, volumes 1 to 3, Cold Spring Harbor Laboratory Press (1989)! was grown at 37.degree. C. in 10 ml LB medium until the optical absorbance at 600 nm reached the value of 0.5. The culture was infected with 650 microliters of phage eluted from the magnetic beads and briefly incubated at 37.degree. C. with no shaking. After centrifugation, the infected cells were resuspended in 2 ml LB medium and plated onto 245.times.245.times.25 mm plates filled with LB-agar containing 1% glucose and 100 micrograms/ml carbenicillin. After overnight incubation at 37.degree. C., the cells were scraped from the plates and resuspended in 40 ml LB medium supplemented with 1% glucose and 100 micrograms/ml carbenicillin. A cell aliquot corresponding to 15 optical densities at 600 nm was then used to inoculate 100 ml LB medium containing 1% glucose and 100 micrograms/ml carbenicillin. Phage rescue for the next panning round was done as outlined above.
For the second panning round, 6.times.10.sup.12 phage were incubated during 90 minutes with 1 mg blocked streptavidin-coated magnetic beads in 200 microliters of TBS containing 2.5 mM Ca.sup.2+ and 2% skim milk (this step was introduced in the procedure to avoid selection of streptavidin-binding clones). After removal of the beads, the same protocol was followed as for round 1. Rounds 3, 4 and 5 were accomplished as round 2, except that the phage input was lowered to 2'10.sup.12 phage.
Twenty-four individual carbenicillin resistant clones that were isolated after five rounds of panning against biotinylated factor Xa, were then analysed by ELISA. Streptavidin-coated 96-well plates (Pierce) were blocked for 1 hour with 200 microliters of TBS containing 2% skim milk per well, then were incubated for 1 hour with 100 microliters of 20 nM biotinylated factor Xa in TBS per well. For each clone, about 10.sup.10 phage diluted in 100 microliters TBS containing 2% skim milk and 0.1% Tween-20 were added to the wells. After a 2-hour incubation, the wells were rinsed four times with 200 microliters TBS containing 0.1% Tween-20. Bound phage were visualized by consecutively incubating with a rabbit anti-M13 anti-serum (see Example 11), an alkaline phosphatase conjugated antirabbit serum (Sigma), and p-nitrophenylphosphate as substrate (Sigma). Absorbances were taken at 405 nm after 20 minutes. Out of the 24 clones, five bound strongly to factor Xa. No significant non-specific binding was observed with these phage when tested in the same ELISA with omission of biotinylated factor Xa.
Single stranded DNA was then prepared from the five positive clones and the inserts 3' to the gene 6 were submitted to automated DNA sequencing using the primer #1224 having the sequence, CGCCAGGGTT TTCCCAGTCA CGAC �SEQ. ID. NO. 31! (New England Biolabs). All five clones were found to contain the same 470 bp 5'-truncated cDNA fused in frame to gene 6 in pDONG63. The nucleotide sequence of this cDNA as well as the deduced amino acid sequence are depicted in FIG. 9. The cDNA, designated NAPc2, encodes a protein, designated NAP isoform c2, that belongs to the NAP family of related proteins.
Example 11
Preparation of Antiserum Aaainst M13 Phage.
Antiserum against M13 phage was prepared in rabbits by subcutaneous injections of about 10.sup.13 M13KO7 phage in 500 microliters of PBS (0.01M sodium phosphate, pH 7.4+0.15M sodium chloride) combined with an equal volume of adjuvant. The M13KO7 phage were CsCl-purified essentially as described by Glaser-Wuttke, G., Keppner, J., and Rasched, I., Biochim. Biophys. Acta, 985: 239-247 (1989). The initial injection was done with Complete Freunds adjuvant on day 0, followed by subsequent injections with Incomplete Freunds adjuvant on days 7, 14 and 35. Antiserum was harvested on day 42.
The IgG fraction of the antiserum was enriched by passage over a Protein A-Sepharose column using conditions well known in the art.
Example A
Factor Xa Amidolytic Assay.
The ability of NAPs of the present invention to act a inhibitors of factor Xa catalytic activity was assessed by determining the inhibition of amidolytic activity catalyzed by the human enzyme.
The buffer used for all assays was HBSA (10 mM HEPES, pH 7.5, 150 mM sodium chloride, 0.1% bovine serum albumin).
The assay was conducted by combining in appropriate wells of a Corning microliter plate, 50 microliters of HBSA, 50 microliters of the test compound diluted in HBSA (or HBSA alone for uninhibited velocity measurement), and 50 microliters of the enzyme diluted in HBSA (prepared from purified human factor X obtained from Enzyme Research Laboratories according to the method described by Bock, P. E. et al., Archives of Biochem. Biophys. 273: 375 (1989). The enzyme was diluted into HBSA prior to the assay in which the final concentration was 0.5 nM). Following a 30 minute incubation at ambient temperature, 50 microliters of the substrate S2765 (N-alpha-benzyloxycarbonyl-D-argininyl-L-glycyl-L-arginine-p-nitroanilide dihydrochloride, obtained from Kabi Diagnostica and made up in deionized water followed by dilution in HBSA prior to the assay) was added to the wells yielding a final total volume of 200 microliters and a final concentration of 250 micromolar (about 5-times Km). The initial velocity of chromogenic substrate hydrolysis was measured by the change in absorbance at 405 nm using a Thermo Max.RTM. Kinetic Microplate Reader over a 5 minute period in which less than 5% of the added substrate was utilized.
Example B
Prothrombin Time (PT) and activated Partial Thromboplastin Time (aPPT) Assays.
The ex vivo anticoagulant effects of NAP, Pro-NAP isoform 5 and NAP isoform 5 in human plasma was evaluated by measuring the prolongation of the activated partial thromboplastin time (aPTT) and prothrombin time (PT) over a broad concentration range of each inhibitor.
Fresh frozen pooled normal human plasma was obtained from George King Biomedical, Overland Park, Kans. Respective measurements of aPTT and PT were made using the Coag-A-Mate RA4 automated coagulometer (General Diagnostics, Organon Technica, Oklahoma City, Okla.) using the Automated aPTT Platelin.RTM. L reagent (Organon Technica, Durham, N.C.) and Simplastlin.RTM. Excel (Organon Technica, Durham, N.C.) respectively, as initiators of clotting according to the manufacturers instructions.
The assay was conducted by making a series of dilutions of each inhibitor in rapidly thawed plasma followed by adding 200 microliters or 100 microliters to the wells of the assay carousel for the aPTT or PT measurements, respectively. As shown in FIGS. 10A and 10B the inhibitors prolonged the PT (FIG. 10A) and aPTT (FIG. 10B) in a dose-dependent manner.
Table 1 shows the concentrations of the NAPs of the present invention at which the PT or aPTT clotting times were doubled relative to a control assay where no such NAP was present.
TABLE 1______________________________________ Concentration, nMInhibitor PT Assay aPPT Assay______________________________________NAP, native protein 26.9 76.2NAP, isoform 5.sup.a 39.2 60.0Pro-NAP, isoform 5.sup.b 21.9 31.0______________________________________ .sup.a This was made as a recombinant protein in Pichia pastoris cells. .sup.b This was made as a recombinant protein in COS cells.
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 86- (2) INFORMATION FOR SEQ ID NO: 1:- (i) SEQUENCE CHARACTERISTICS:# 33 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:- (ix) FEATURE:#Base (A) NAME/KEY: Modified (B) LOCATION: 6...6#N is i (D) OTHER INFORMATION:#Base (A) NAME/KEY: Modified (B) LOCATION: 12...12#N is i (D) OTHER INFORMATION:#Base (A) NAME/KEY: Modified (B) LOCATION: 21...21#N is i (D) OTHER INFORMATION:# 1: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# 33 GYGG NGARAAYGAR TGG- (2) INFORMATION FOR SEQ ID NO: 2:- (i) SEQUENCE CHARACTERISTICS:# 234 base pairsH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 2: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- AAGGCATACC CGGAGTGTGG TGAGAATGAA TGGCTCGACG ACTGTGGAAC TC - #AGAAGCCA 60- TGCGAGGCCA AGTGCAATGA GGAACCCCCT GAGGAGGAAG ATCCGATATG CC - #GCTCACGT 120- GGTTGTTTAT TACCTCCTGC TTGCGTATGC AAAGACGGAT TCTACAGAGA CA - #CGGTGATC 180- GGCGACTGTG TTAGGGAAGA AGAATGCGAC CAACATGAGA TTATACATGT CT - #GA 234- (2) INFORMATION FOR SEQ ID NO: 3:- (i) SEQUENCE CHARACTERISTICS:# 228 base pairsH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 3: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- AAGGCATACC CGGAGTGTGG TGAGAATGAA TGGCTCGACG TCTGTGGAAC TA - #AGAAGCCA 60- TGCGAGGCCA AGTGCAGTGA GGAAGAGGAG GAAGATCCGA TATGCCGATC AT - #TTTCTTGT 120- CCGGGTCCCG CTGCTTGCGT ATGCGAAGAC GGATTCTACA GAGACACGGT GA - #TCGGCGAC 180# 228AATG CGACCAACAT GAGATTATAC ATGTCTGA- (2) INFORMATION FOR SEQ ID NO: 4:- (i) SEQUENCE CHARACTERISTICS:# 6 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:#4: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Gly Gly Gly Ser Gly Gly 1 5- (2) INFORMATION FOR SEQ ID NO: 5:- (i) SEQUENCE CHARACTERISTICS:# 9 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2 to 9ION:#amino acid. is an#5: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5- (2) INFORMATION FOR SEQ ID NO: 6:- (i) SEQUENCE CHARACTERISTICS:# 13 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: N-terminal fragme - #nt#6: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Ala Tyr Pro Glu Cys Gly Glu Asn Glu Tr - #p Leu Asp# 10- (2) INFORMATION FOR SEQ ID NO: 7:- (i) SEQUENCE CHARACTERISTICS:# 11 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:#7: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Ala Tyr Pro Glu Cys Gly Glu Asn Glu Tr - #p# 10- (2) INFORMATION FOR SEQ ID NO: 8:- (i) SEQUENCE CHARACTERISTICS:# 28 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 8: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# 28 TTTT TTTTTTTT- (2) INFORMATION FOR SEQ ID NO: 9:- (i) SEQUENCE CHARACTERISTICS:# 24 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 9: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# 24GGAG CCCG- (2) INFORMATION FOR SEQ ID NO: 10:- (i) SEQUENCE CHARACTERISTICS:# 20 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:#10: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Ala Tyr Pro Glu Cys Gly Glu Asn Glu Tr - #p Leu Asp Asp Cys GlyThr# 15- Gln Lys Pro 20- (2) INFORMATION FOR SEQ ID NO: 11:- (i) SEQUENCE CHARACTERISTICS:# 10 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:#11: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# 10- (2) INFORMATION FOR SEQ ID NO: 12:- (i) SEQUENCE CHARACTERISTICS:# 18 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:#12: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# 18 GT- (2) INFORMATION FOR SEQ ID NO: 13:- (i) SEQUENCE CHARACTERISTICS:# 18 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:#13: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# 18 GG- (2) INFORMATION FOR SEQ ID NO: 14:- (i) SEQUENCE CHARACTERISTICS:# 24 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 14: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# 24ACAC AGGA- (2) INFORMATION FOR SEQ ID NO: 15:- (i) SEQUENCE CHARACTERISTICS:# 66 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 15: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- ATGTTCTCTC CAATTTTGTC CTTGGAAATT ATTTTAGCTT TGGCTACTTT GC - #AATCTGTC 60# 66- (2) INFORMATION FOR SEQ ID NO: 16:- (i) SEQUENCE CHARACTERISTICS:# 57 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 16: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- CAGCCAGGTA TCTCCACTAC CGTTGGTTCC GCTGCCGAGG GTTCTTTGGA CA - #AGAGG 57- (2) INFORMATION FOR SEQ ID NO: 17:- (i) SEQUENCE CHARACTERISTICS:# 51 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 17: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# 51TTCAGAT CTGAATGCGG CCGCTCGAGA CTAGTGGATC C- (2) INFORMATION FOR SEQ ID NO: 18:- (i) SEQUENCE CHARACTERISTICS:# 41 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 18: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# 41 TCAG ACATGTATAA TCTCATGTTG G- (2) INFORMATION FOR SEQ ID NO: 19:- (i) SEQUENCE CHARACTERISTICS:# 22 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 19: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# 22TGG TG- (2) INFORMATION FOR SEQ ID NO: 20:- (i) SEQUENCE CHARACTERISTICS:# 5 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: N-terminal fragme - #nt#20: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Ala Tyr Pro Glu 1 5- (2) INFORMATION FOR SEQ ID NO: 21:- (i) SEQUENCE CHARACTERISTICS:# 36 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 21: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# 36 GAAG ATGCTTTACG CTATCG- (2) INFORMATION FOR SEQ ID NO: 22:- (i) SEQUENCE CHARACTERISTICS:# 23 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 22: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# 23TCTC AAG- (2) INFORMATION FOR SEQ ID NO: 23:- (i) SEQUENCE CHARACTERISTICS:# 9 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: N-terminal fragme - #nt#23: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Arg Thr Val Arg Lys Ala Tyr Pro Glu 1 5- (2) INFORMATION FOR SEQ ID NO: 24:- (i) SEQUENCE CHARACTERISTICS:# 5 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: N-terminal fragme - #nt#24: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Arg Thr Val Arg Lys 1 5- (2) INFORMATION FOR SEQ ID NO: 25:- (i) SEQUENCE CHARACTERISTICS:# 25 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 25: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# 25 TCAT GTTGG- (2) INFORMATION FOR SEQ ID NO: 26:- (i) SEQUENCE CHARACTERISTICS:# 33 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 26: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# 33 ACAT ACTGCGTAAT AAG- (2) INFORMATION FOR SEQ ID NO: 27:- (i) SEQUENCE CHARACTERISTICS:# 60 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 27: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- TATGGGATGG CCGACTTGGC CTCCGCCTGA GCCTCCACCT TTATCCCAAT CC - #AAATAAGA 60- (2) INFORMATION FOR SEQ ID NO: 28:- (i) SEQUENCE CHARACTERISTICS:# 60 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 28: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- ATGGGATGGC CGACTTGGCC CTCCGCCTGA GCCTCCACCT TTATCCCAAT CC - #AAATAAGA 60- (2) INFORMATION FOR SEQ ID NO: 29:- (i) SEQUENCE CHARACTERISTICS:# 60 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 29: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- TATGGGATGG CCGACTTGGC CGATCCGCCT GAGCCTCCAC CTTTATCCCA AT - #CCAAATAA 60- (2) INFORMATION FOR SEQ ID NO: 30:- (i) SEQUENCE CHARACTERISTICS:# 45 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 30: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:#45 CCGC GTGATATGGG ATGGCCGACT TGGCC- (2) INFORMATION FOR SEQ ID NO: 31:- (i) SEQUENCE CHARACTERISTICS:# 24 base pairsTH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 31: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:# 24GTCA CGAC- (2) INFORMATION FOR SEQ ID NO: 32:- (i) SEQUENCE CHARACTERISTICS:# 461 base pairsH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:- (ix) FEATURE: (A) NAME/KEY: Coding Se - #quence (B) LOCATION: 22...321 (D) OTHER INFORMATION:#32: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:#ATC GCT ATA ATG 51 ATG AAG ATG CTT TAC GCT#Ala Ile Metys Met Leu Tyr Ala Ile# 10- TTT CTC CTG GTA TCA TTA TGC AGC GCA AGA AC - #A GTG AGG AAG GCA TAC 99Phe Leu Leu Val Ser Leu Cys Ser Ala Arg Th - #r Val Arg Lys Ala Tyr# 25- CCG GAG TGT GGT GAG AAT GAA TGG CTC GAC GA - #C TGT GGA ACT CAG AAG 147Pro Glu Cys Gly Glu Asn Glu Trp Leu Asp As - #p Cys Gly Thr Gln Lys# 40- CCA TGC GAG GCC AAG TGC AAT GAG GAA CCC CC - #T GAG GAG GAA GAT CCG 195Pro Cys Glu Ala Lys Cys Asn Glu Glu Pro Pr - #o Glu Glu Glu Asp Pro# 55- ATA TGC CGC TCA CGT GGT TGT TTA TTA CCT CC - #T GCT TGC GTA TGC AAA 243Ile Cys Arg Ser Arg Gly Cys Leu Leu Pro Pr - #o Ala Cys Val Cys Lys# 70- GAC GGA TTC TAC AGA GAC ACG GTG ATC GGC GA - #C TGT GTT AGG GAA GAA 291Asp Gly Phe Tyr Arg Asp Thr Val Ile Gly As - #p Cys Val Arg Glu Glu#90- GAA TGC GAC CAA CAT GAG ATT ATA CAT GTC TG - #AACGAGAA AGCAACAATA ACCA 345Glu Cys Asp Gln His Glu Ile Ile His Val# 100- AAGGTTCCAA CTCTCGCTCT GCAAAATCGC TAGTTGGATG TCTCTTTTGC GT - #CCGAATAG 405- TTTTAGTTGA TGTTAAGTAA GAACTCCTGC TGGAGAGAAT AAAGCTTTCC AA - #CTCC 461- (2) INFORMATION FOR SEQ ID NO: 33:- (i) SEQUENCE CHARACTERISTICS:# 77 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:#33: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Ala Tyr Pro Glu Cys Gly Glu Asn Glu Tr - #p Leu Asp Asp Cys Gly ThrGln# 15- Lys Pro Cys Glu Ala Lys Cys Asn Glu Glu Pr - #o Pro Glu Glu Glu Asp ProIle# 350- Cys Arg Ser Arg Gly Cys Leu Leu Pro Pro Al - #a Cys Val Cys Lys Asp GlyPhe# 50- Tyr Arg Asp Thr Val Ile Gly Asp Cys Val Ar - #g Glu Glu Glu Cys Asp GlnHis# 70- Glu Ile Ile His Val 75- (2) INFORMATION FOR SEQ ID NO: 34:- (i) SEQUENCE CHARACTERISTICS:# 455 base pairsH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:- (ix) FEATURE: (A) NAME/KEY: Coding Se - #quence (B) LOCATION: 22...315 (D) OTHER INFORMATION:# 34: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:#ATC GCT ATA ATG 51 ATG AAG ATG CTT TAC GCT#Ala Ile Metys Met Leu Tyr Ala Ile# 10- TTT CTC CTG GTG TCA TTA TGC AGC ACA AGA AC - #A GTG AGG AAG GCA TAC 99Phe Leu Leu Val Ser Leu Cys Ser Thr Arg Th - #r Val Arg Lys Ala Tyr# 25- CCG GAG TGT GGT GAG AAT GAA TGG CTC GAC GT - #C TGT GGA ACT AAG AAG 147Pro Glu Cys Gly Glu Asn Glu Trp Leu Asp Va - #l Cys Gly Thr Lys Lys# 40- CCA TGC GAG GCC AAG TGC AGT GAG GAA GAG GA - #G GAA GAT CCG ATA TGC 195Pro Cys Glu Ala Lys Cys Ser Glu Glu Glu Gl - #u Glu Asp Pro Ile Cys# 55- CGA TCA TTT TCT TGT CCG GGT CCC GCT GCT TG - #C GTA TGC GAA GAC GGA 243Arg Ser Phe Ser Cys Pro Gly Pro Ala Ala Cy - #s Val Cys Glu Asp Gly# 70- TTC TAC AGA GAC ACG GTG ATC GGC GAC TGT GT - #T AAG GAA GAA GAA TGC 291Phe Tyr Arg Asp Thr Val Ile Gly Asp Cys Va - #l Lys Glu Glu Glu Cys#90- GAC CAA CAT GAG ATT ATT CAT GTC TGAACGAGAG AG - #CAGTAATA ACCAAAGGTT C 346Asp Gln His Glu Ile Ile His Val 95- CAACTTTCGC TCTACAAAAT CGCTAGTTGG ATTTCTCCTT TGCGTGCGAA TA - #GTTTTAGT 406# 455CCTCC TGTTGAAGAG AATAAAGCTT TCCAACTTC- (2) INFORMATION FOR SEQ ID NO: 35:- (i) SEQUENCE CHARACTERISTICS:# 75 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:# 35: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Ala Tyr Pro Glu Cys Gly Glu Asn Glu Tr - #p Leu Asp Val Cys Gly ThrLys# 15- Lys Pro Cys Glu Ala Lys Cys Ser Glu Glu Gl - #u Glu Glu Asp Pro Ile CysArg# 35- Ser Phe Ser Cys Pro Gly Pro Ala Ala Cys Va - #l Cys Glu Asp Gly Phe TyrArg# 50- Asp Thr Val Ile Gly Asp Cys Val Lys Glu Gl - #u Glu Cys Asp Gln His GluIle# 70- Ile His Val- (2) INFORMATION FOR SEQ ID NO: 36:- (i) SEQUENCE CHARACTERISTICS:# 81 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:# 36: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Arg Thr Val Arg Lys Ala Tyr Pro Glu Cys Gl - #y Glu Asn Glu Trp Leu AspAsp# 15- Cys Gly Thr Gln Lys Pro Cys Glu Ala Lys Cy - #s Asn Glu Glu Pro Pro GluGlu# 35- Glu Asp Pro Ile Cys Arg Ser Arg Gly Cys Le - #u Leu Pro Pro Ala Cys ValCys# 50- Lys Asp Gly Phe Tyr Arg Asp Thr Val Ile Gl - #y Asp Cys Val Arg Glu GluGlu# 70- Cys Asp Gln His Glu Ile Ile His Val# 80- (2) INFORMATION FOR SEQ ID NO: 37:- (i) SEQUENCE CHARACTERISTICS:# 79 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:# 37: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Arg Thr Val Arg Lys Ala Tyr Pro Glu Cys Gl - #y Glu Asn Glu Trp Leu AspVal# 15- Cys Gly Thr Lys Lys Pro Cys Glu Ala Lys Cy - #s Ser Glu Glu Glu Glu GluAsp# 35- Pro Ile Cys Arg Ser Phe Ser Cys Pro Gly Pr - #o Ala Ala Cys Val Cys GluAsp# 50- Gly Phe Tyr Arg Asp Thr Val Ile Gly Asp Cy - #s Val Lys Glu Glu Glu CysAsp# 70- Gln His Glu Ile Ile His Val 75- (2) INFORMATION FOR SEQ ID NO: 38:- (i) SEQUENCE CHARACTERISTICS:# 711 base pairsH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:- (ix) FEATURE: (A) NAME/KEY: Coding Se - #quence (B) LOCATION: 21...590 (D) OTHER INFORMATION:#38: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GAATTCACTA TTATCCAACA ATG GCG GTG CTT TAT TCA GT - #A GCA ATA GCG 50#Ile Ala Ala Val Leu Tyr Ser Val Ala# 10- TTA CTA CTG GTA TCA CAA TGC AGT GGG AAA CC - #G AAC AAT GTG ATG ACT 98Leu Leu Leu Val Ser Gln Cys Ser Gly Lys Pr - #o Asn Asn Val Met Thr# 25- AAC GCT TGT GGT CTT AAT GAA TAT TTC GCT GA - #G TGT GGC AAT ATG AAG 146Asn Ala Cys Gly Leu Asn Glu Tyr Phe Ala Gl - #u Cys Gly Asn Met Lys# 40- GAA TGC GAG CAC AGA TGC AAT GAG GAG GAA AA - #T GAG GAA AGG GAC GAG 194Glu Cys Glu His Arg Cys Asn Glu Glu Glu As - #n Glu Glu Arg Asp Glu# 55- GAA AGA ATA ACG GCA TGC CTC ATC CGT GTG TG - #T TTC CGT CCT GGT GCT 242Glu Arg Ile Thr Ala Cys Leu Ile Arg Val Cy - #s Phe Arg Pro Gly Ala# 70- TGC GTA TGC AAA GAC GGA TTC TAT AGA AAC AG - #A ACA GGC AGC TGT GTG 290Cys Val Cys Lys Asp Gly Phe Tyr Arg Asn Ar - #g Thr Gly Ser Cys Val#90- GAA GAA GAT GAC TGC GAG TAC GAG AAT ATG GA - #G TTC ATT ACT TTT GCA 338Glu Glu Asp Asp Cys Glu Tyr Glu Asn Met Gl - #u Phe Ile Thr Phe Ala# 105- CCA GAA GTA CCG ATA TGT GGT TCC AAC GAA AG - #G TAC TCC GAC TGC GGC 386Pro Glu Val Pro Ile Cys Gly Ser Asn Glu Ar - #g Tyr Ser Asp Cys Gly# 120- AAT GAC AAA CAA TGC GAG CGC AAA TGC AAC GA - #G GAC GAT TAT GAG AAG 434Asn Asp Lys Gln Cys Glu Arg Lys Cys Asn Gl - #u Asp Asp Tyr Glu Lys# 135- GGA GAT GAG GCA TGC CGC TCA CAT GTT TGT GA - #A CGT CCT GGT GCC TGT 482Gly Asp Glu Ala Cys Arg Ser His Val Cys Gl - #u Arg Pro Gly Ala Cys# 150- GTA TGC GAA GAC GGG TTC TAC AGA AAC AAA AA - #A GGT AGC TGT GTG GAA 530Val Cys Glu Asp Gly Phe Tyr Arg Asn Lys Ly - #s Gly Ser Cys Val Glu155 1 - #60 1 - #65 1 -#70- AGC GAT GAC TGC GAA TAC GAT AAT ATG GAT TT - #C ATC ACT TTT GCA CCA 578Ser Asp Asp Cys Glu Tyr Asp Asn Met Asp Ph - #e Ile Thr Phe Ala Pro# 185- GAA ACC TCA CGA TAACCAAAGA TGCTACCTCT CGTACGCAAC TC - #CGCTGATT GAGGTT 636Glu Thr Ser Arg 190- GATTCACTCC CTTGCATCTC AACATTTTTT TTGTGATGCT GTGCATCTGA GC - #TTAACCTG 696# 711- (2) INFORMATION FOR SEQ ID NO: 39:- (i) SEQUENCE CHARACTERISTICS:# 425 base pairsH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:- (ix) FEATURE: (A) NAME/KEY: Coding Se - #quence (B) LOCATION: 10...291 (D) OTHER INFORMATION:# 39: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GAATTCCGC ATG CGG ACG CTC TAC CTC ATT TCT ATC - # TGG TTG TTC CTC ATC 51#Leu Ile Ser Ile Trp Leu Phe Leu Ile# 10- TCG CAA TGT AAT GGA AAA GCA TTC CCG AAA TG - #T GAC GTC AAT GAA AGA 99Ser Gln Cys Asn Gly Lys Ala Phe Pro Lys Cy - #s Asp Val Asn Glu Arg#30- TTC GAG GTG TGT GGC AAT CTG AAG GAG TGC GA - #G CTC AAG TGC GAT GAG 147Phe Glu Val Cys Gly Asn Leu Lys Glu Cys Gl - #u Leu Lys Cys Asp Glu# 45- GAC CCT AAG ATA TGC TCT CGT GCA TGT ATT CG - #T CCC CCT GCT TGC GTA 195Asp Pro Lys Ile Cys Ser Arg Ala Cys Ile Ar - #g Pro Pro Ala Cys Val# 60- TGC GAT GAC GGA TTC TAC AGA GAC AAA TAT GG - #C TTC TGT GTT GAA GAA 243Cys Asp Asp Gly Phe Tyr Arg Asp Lys Tyr Gl - #y Phe Cys Val Glu Glu# 75- GAC GAA TGT AAC GAT ATG GAG ATT ATT ACT TT - #T CCA CCA GAA ACC AAA TG 293Asp Glu Cys Asn Asp Met Glu Ile Ile Thr Ph - #e Pro Pro Glu Thr Lys# 90- ATGACCGAAG CTTCCACCTT TCTATACATA TCTTCACTGC TTGACAGGCT TC - #TCGACAAT 353- TTAGAAGTTC TGCTTGACTT TGTCTATTTG AAATTGTTCA CACTAATGGG GG - #AAGTAAAG 413# 425- (2) INFORMATION FOR SEQ ID NO: 40:- (i) SEQUENCE CHARACTERISTICS:# 471 base pairsH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:- (ix) FEATURE: (A) NAME/KEY: Coding Se - #quence (B) LOCATION: 23...310 (D) OTHER INFORMATION:# 40: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GAATTCCGCT ACATTTTCAA CA ATG TCG ACG CTT TAT GTT - # ATC GCA ATA TGT 52# Met Ser Thr Leu Tyr Val I - #le Ala Ile Cys# 10- TTG CTG CTT GTT TCG CAA TGC AAT GGA AGA AC - #G GTG AAG AAG TGT GGC 100Leu Leu Leu Val Ser Gln Cys Asn Gly Arg Th - #r Val Lys Lys Cys Gly# 25- AAG AAT GAA AGA TAC GAC GAC TGT GGC AAT GC - #A AAG GAC TGC GAG ACC 148Lys Asn Glu Arg Tyr Asp Asp Cys Gly Asn Al - #a Lys Asp Cys Glu Thr# 40- AAG TGC GGT GAA GAG GAA AAG GTG TGC CGT TC - #G CGT GAG TGT ACT AGT 196Lys Cys Gly Glu Glu Glu Lys Val Cys Arg Se - #r Arg Glu Cys Thr Ser# 55- CCT GGT GCC TGC GTA TGC GAA CAA GGA TTC TA - #C AGA GAT CCG GCT GGC 244Pro Gly Ala Cys Val Cys Glu Gln Gly Phe Ty - #r Arg Asp Pro Ala Gly# 70- GAC TGT GTC ACT GAT GAA GAA TGT GAT GAA TG - #G AAC AAT ATG GAG ATC 292Asp Cys Val Thr Asp Glu Glu Cys Asp Glu Tr - #p Asn Asn Met Glu Ile#90- ATT ACT ATG CCA AAA CAG TAGTGCGAAG TTCCCTTCTT TC - #TCCAAATC TGCTCCGTG 349Ile Thr Met Pro Lys Gln 95- CTCAATTATC ACACACCTCC ACTAGTTAAG ATTGACTGAC TCTCTTGCAT TG - #TAGTATTT 409- TCGCTTGACT CTGTGCATTT AAGCATGAGA TACTACTAGG GAGAATAAAA AT - #TACTAACT 469# 471- (2) INFORMATION FOR SEQ ID NO: 41:- (i) SEQUENCE CHARACTERISTICS:# 396 base pairsH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:- (ix) FEATURE: (A) NAME/KEY: Coding Se - #quence (B) LOCATION: 10...237 (D) OTHER INFORMATION:# 41: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GAATTCCGG AAA TGT CCT ACC GAT GAA TGG TTC GAT - # TGG TGT GGA ACT TAC 51#Glu Trp Phe Asp Trp Cys Gly Thr Tyr# 10- AAG CAT TGC GAA CTC AAG TGC GAT AGG GAG CT - #A ACT GAG AAA GAA GAG 99Lys His Cys Glu Leu Lys Cys Asp Arg Glu Le - #u Thr Glu Lys Glu Glu#30- CAG GCA TGT CTC TCA CGT GTT TGT GAG AAG TC - #C GCT TGC GTA TGC AAT 147Gln Ala Cys Leu Ser Arg Val Cys Glu Lys Se - #r Ala Cys Val Cys Asn# 45- GAC GGA TTA TAC AGA GAC AAG TTT GGC AAC TG - #T GTT GAA AAA GAC GAA 195Asp Gly Leu Tyr Arg Asp Lys Phe Gly Asn Cy - #s Val Glu Lys Asp Glu# 60- TGC AAC GAT ATG GAG ATT ATT ACT TTT GCA CC - #A GAA ACC AAA TAATGGCCTA 247Cys Asn Asp Met Glu Ile Ile Thr Phe Ala Pr - #o Glu Thr Lys# 75- AGGTTCCAAA CCTTGCTACA CACCGTCAGT GCTTTACTGT TTCCTCTACG TG - #TTAGTAGT 307- TTTGCTTGAC TCTGTGTATT TAAGCATTGT CTACTAATGG GCAAAGTAAA GC - #ATTGTAAG 367# 396 AACC TTCTGATTT- (2) INFORMATION FOR SEQ ID NO: 42:- (i) SEQUENCE CHARACTERISTICS:# 688 base pairsH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:- (ix) FEATURE: (A) NAME/KEY: Coding Se - #quence (B) LOCATION: 21...560 (D) OTHER INFORMATION:# 42: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GAATTCCGGG CGGCAGAAAG ATG CGA ATG CTC TAC CTT GT - #T CCT ATC TGG 50#Ile Trp Arg Met Leu Tyr Leu Val Pro# 10- TTG CTG CTC ATT TCG CTA TGC AGT GGA AAA GC - #T GCG AAG AAA TGT GGT 98Leu Leu Leu Ile Ser Leu Cys Ser Gly Lys Al - #a Ala Lys Lys Cys Gly# 25- CTC AAT GAA AGG CTG GAC TGT GGC AAT CTG AA - #G CAA TGC GAG CCC AAG 146Leu Asn Glu Arg Leu Asp Cys Gly Asn Leu Ly - #s Gln Cys Glu Pro Lys# 40- TGC AGC GAC TTG GAA AGT GAG GAG TAT GAG GA - #G GAA GAT GAG TCG AAA 194Cys Ser Asp Leu Glu Ser Glu Glu Tyr Glu Gl - #u Glu Asp Glu Ser Lys# 55- TGT CGA TCA CGT GAA TGT TCT CGT CGT GTT TG - #T GTA TGC GAT GAA GGA 242Cys Arg Ser Arg Glu Cys Ser Arg Arg Val Cy - #s Val Cys Asp Glu Gly# 70- TTC TAC AGA AAC AAG AAG GGC AAG TGT GTT GC - #A AAA GAT GTT TGC GAG 290Phe Tyr Arg Asn Lys Lys Gly Lys Cys Val Al - #a Lys Asp Val Cys Glu#90- GAC GAC AAT ATG GAG ATT ATC ACT TTT CCA CC - #A GAA GAC GAA TGT GGT 338Asp Asp Asn Met Glu Ile Ile Thr Phe Pro Pr - #o Glu Asp Glu Cys Gly# 105- CCC GAT GAA TGG TTC GAC TAC TGT GGA AAT TA - #T AAG AAG TGC GAA CGC 386Pro Asp Glu Trp Phe Asp Tyr Cys Gly Asn Ty - #r Lys Lys Cys Glu Arg# 120- AAG TGC AGT GAG GAG ACA AGT GAG AAA AAT GA - #G GAG GCA TGC CTC TCT 434Lys Cys Ser Glu Glu Thr Ser Glu Lys Asn Gl - #u Glu Ala Cys Leu Ser# 135- CGT GCT TGT ACT GGT CGT GCT TGC GTA TGC AA - #A GAC GGA TTG TAC AGA 482Arg Ala Cys Thr Gly Arg Ala Cys Val Cys Ly - #s Asp Gly Leu Tyr Arg# 150- GAC GAC TTT GGC AAC TGT GTT CCA CAT GAC GA - #A TGC AAC GAT ATG GAG 530Asp Asp Phe Gly Asn Cys Val Pro His Asp Gl - #u Cys Asn Asp Met Glu155 1 - #60 1 - #65 1 -#70- ATC ATC ACT TTT CCA CCG GAA ACC AAA CAT TG - #ACCAGAGG CTCCAACTCT CGCT 584Ile Ile Thr Phe Pro Pro Glu Thr Lys His# 180- ACACAACGTC AGGGCTAGAA TGGCCCCTCT GCGAGTTAGT AGTTTTGCTT GA - #CTCTGCTT 644#688 TTGA TGGCGAAAAT AAAGCATTTA AAAC- (2) INFORMATION FOR SEQ ID NO: 43:- (i) SEQUENCE CHARACTERISTICS:# 349 base pairsH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:- (ix) FEATURE: (A) NAME/KEY: Coding Se - #quence (B) LOCATION: 49...276 (D) OTHER INFORMATION:# 43: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:#ATC CGA 57CTGAGAG GTGAGCTACG CAAGTCTTCG CTGGTACA ATG# Met - # Ile Arg#1- AAG CTC GTT CTG CTG ACT GCT ATC GTC ACG GT - #G GTG CTA AGT GCG AAG 105Lys Leu Val Leu Leu Thr Ala Ile Val Thr Va - #l Val Leu Ser Ala Lys# 15- ACC TGT GGA CCA AAC GAG GAG TAC ACT GAA TG - #C GGG ACG CCA TGC GAG 153Thr Cys Gly Pro Asn Glu Glu Tyr Thr Glu Cy - #s Gly Thr Pro Cys Glu#35- CCG AAG TGC AAT GAA CCG ATG CCA GAC ATC TG - #T ACT CTG AAC TGC ATC 201Pro Lys Cys Asn Glu Pro Met Pro Asp Ile Cy - #s Thr Leu Asn Cys Ile# 50- GTG AAC GTG TGT CAG TGC AAA CCC GGC TTC AA - #G CGC GGA CCG AAA GGA 249Val Asn Val Cys Gln Cys Lys Pro Gly Phe Ly - #s Arg Gly Pro Lys Gly# 65- TGC GTC GCC CCC GGA CCA GGC TGT AAA TAGTTCTCC - #A CCTGCCCTTT CGTTGGAA 304Cys Val Ala Pro Gly Pro Gly Cys Lys# 75# 349CA TTCTGAATCA ATAAAGCCGA ACGGT- (2) INFORMATION FOR SEQ ID NO: 44:- (i) SEQUENCE CHARACTERISTICS:# 432 base pairsH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:- (ix) FEATURE: (A) NAME/KEY: Coding Se - #quence (B) LOCATION: 40...393 (D) OTHER INFORMATION:# 44: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- AAGCTTTGCT AACATACTGC GTAATAAGGA GTCTTAATC ATG CCA GTT - # CTT TTG 54# Met Pro Val Leu Leu# 5 1- GGT ATT CCG TTA TTA TTG CGT TTC CTC GGT TT - #C CTT CTG GTA ACT TTG 102Gly Ile Pro Leu Leu Leu Arg Phe Leu Gly Ph - #e Leu Leu Val Thr Leu# 20- TTC GGC TAT CTG CTT ACT TTC CTT AAA AAG GG - #C TTC GGT AAG ATA GCT 150Phe Gly Tyr Leu Leu Thr Phe Leu Lys Lys Gl - #y Phe Gly Lys Ile Ala# 35- ATT GCT ATT TCA TTG TTT CTT GCT CTT ATT AT - #T GGG CTT AAC TCA ATT 198Ile Ala Ile Ser Leu Phe Leu Ala Leu Ile Il - #e Gly Leu Asn Ser Ile# 50- CTT GTG GGT TAT CTC TCT GAT ATT AGC GCA CA - #A TTA CCC TCT GAT TTT 246Leu Val Gly Tyr Leu Ser Asp Ile Ser Ala Gl - #n Leu Pro Ser Asp Phe# 65- GTT CAG GGC GTT CAG TTA ATT CTC CCG TCT AA - #T GCG CTT CCC TGT TTT 294Val Gln Gly Val Gln Leu Ile Leu Pro Ser As - #n Ala Leu Pro Cys Phe#85- TAT GTT ATT CTC TCT GTA AAG GCT GCT ATT TT - #C ATT TTT GAC GTT AAA 342Tyr Val Ile Leu Ser Val Lys Ala Ala Ile Ph - #e Ile Phe Asp Val Lys# 100- CAA AAA ATC GTT TCT TAT TTG GAT TGG GAT AA - #A GGT GGA GGC TCA GGC 390Gln Lys Ile Val Ser Tyr Leu Asp Trp Asp Ly - #s Gly Gly Gly Ser Gly# 115# 432 CATCCCAT ATCACGCGGC CGCGGATCCGly- (2) INFORMATION FOR SEQ ID NO: 45:- (i) SEQUENCE CHARACTERISTICS:# 433 base pairsH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:- (ix) FEATURE: (A) NAME/KEY: Coding Se - #quence (B) LOCATION: 40...393 (D) OTHER INFORMATION:# 45: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- AAGCTTTGCT AACATACTGC GTAATAAGGA GTCTTAATC ATG CCA GTT - # CTT TTG 54# Met Pro Val Leu Leu# 5 1- GGT ATT CCG TTA TTA TTG CGT TTC CTC GGT TT - #C CTT CTG GTA ACT TTG 102Gly Ile Pro Leu Leu Leu Arg Phe Leu Gly Ph - #e Leu Leu Val Thr Leu# 20- TTC GGC TAT CTG CTT ACT TTC CTT AAA AAG GG - #C TTC GGT AAG ATA GCT 150Phe Gly Tyr Leu Leu Thr Phe Leu Lys Lys Gl - #y Phe Gly Lys Ile Ala# 35- ATT GCT ATT TCA TTG TTT CTT GCT CTT ATT AT - #T GGG CTT AAC TCA ATT 198Ile Ala Ile Ser Leu Phe Leu Ala Leu Ile Il - #e Gly Leu Asn Ser Ile# 50- CTT GTG GGT TAT CTC TCT GAT ATT AGC GCA CA - #A TTA CCC TCT GAT TTT 246Leu Val Gly Tyr Leu Ser Asp Ile Ser Ala Gl - #n Leu Pro Ser Asp Phe# 65- GTT CAG GGC GTT CAG TTA ATT CTC CCG TCT AA - #T GCG CTT CCC TGT TTT 294Val Gln Gly Val Gln Leu Ile Leu Pro Ser As - #n Ala Leu Pro Cys Phe# 85- TAT GTT ATT CTC TCT GTA AAG GCT GCT ATT TT - #C ATT TTT GAC GTT AAA 342Tyr Val Ile Leu Ser Val Lys Ala Ala Ile Ph - #e Ile Phe Asp Val Lys# 100- CAA AAA ATC GTT TCT TAT TTG GAT TGG GAT AA - #A GGT GGA GGC TCA GGC 390Gln Lys Ile Val Ser Tyr Leu Asp Trp Asp Ly - #s Gly Gly Gly Ser Gly# 115#433 CCATCCCA TATCACGCGG CCGCGGATCCGly- (2) INFORMATION FOR SEQ ID NO: 46:- (i) SEQUENCE CHARACTERISTICS:# 434 base pairsH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:- (ix) FEATURE: (A) NAME/KEY: Coding Se - #quence (B) LOCATION: 40...393 (D) OTHER INFORMATION:# 46: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- AAGCTTTGCT AACATACTGC GTAATAAGGA GTCTTAATC ATG CCA GTT - # CTT TTG 54# Met Pro Val Leu Leu# 5 1- GGT ATT CCG TTA TTA TTG CGT TTC CTC GGT TT - #C CTT CTG GTA ACT TTG 102Gly Ile Pro Leu Leu Leu Arg Phe Leu Gly Ph - #e Leu Leu Val Thr Leu# 20- TTC GGC TAT CTG CTT ACT TTC CTT AAA AAG GG - #C TTC GGT AAG ATA GCT 150Phe Gly Tyr Leu Leu Thr Phe Leu Lys Lys Gl - #y Phe Gly Lys Ile Ala# 35- ATT GCT ATT TCA TTG TTT CTT GCT CTT ATT AT - #T GGG CTT AAC TCA ATT 198Ile Ala Ile Ser Leu Phe Leu Ala Leu Ile Il - #e Gly Leu Asn Ser Ile# 50- CTT GTG GGT TAT CTC TCT GAT ATT AGC GCA CA - #A TTA CCC TCT GAT TTT 246Leu Val Gly Tyr Leu Ser Asp Ile Ser Ala Gl - #n Leu Pro Ser Asp Phe# 65- GTT CAG GGC GTT CAG TTA ATT CTC CCG TCT AA - #T GCG CTT CCC TGT TTT 294Val Gln Gly Val Gln Leu Ile Leu Pro Ser As - #n Ala Leu Pro Cys Phe#85- TAT GTT ATT CTC TCT GTA AAG GCT GCT ATT TT - #C ATT TTT GAC GTT AAA 342Tyr Val Ile Leu Ser Val Lys Ala Ala Ile Ph - #e Ile Phe Asp Val Lys# 100- CAA AAA ATC GTT TCT TAT TTG GAT TGG GAT AA - #A GGT GGA GGC TCA GGC 390Gln Lys Ile Val Ser Tyr Leu Asp Trp Asp Ly - #s Gly Gly Gly Ser Gly# 115#434 GCCATCCC ATATCACGCG GCCGCGGATC CGly- (2) INFORMATION FOR SEQ ID NO: 47:- (i) SEQUENCE CHARACTERISTICS:# 430 base pairsH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:- (ix) FEATURE: (A) NAME/KEY: Coding Se - #quence (B) LOCATION: 10...282 (D) OTHER INFORMATION:# 47: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- GAATTCCGG CTG GTW TCC TAC TGC AGT GGA AAA GCA - # ACG ATG CAG TGT GGT 51#Ser Gly Lys Ala Thr Met Gln Cys Gly# 10- GAG AAT GAA AAG TAC GAT TCG TGC GGT AGC AA - #G GAG TGC GAT AAG AAG 99Glu Asn Glu Lys Tyr Asp Ser Cys Gly Ser Ly - #s Glu Cys Asp Lys Lys#30- TGC AAA TAT GAC GGA GTT GAG GAG GAA GAC GA - #C GAG GAA CCT AAT GTG 147Cys Lys Tyr Asp Gly Val Glu Glu Glu Asp As - #p Glu Glu Pro Asn Val# 45- CCA TGC CTA GTA CGT GTG TGT CAT CAA GAT TG - #C GTA TGC GAA GAA GGA 195Pro Cys Leu Val Arg Val Cys His Gln Asp Cy - #s Val Cys Glu Glu Gly# 60- TTC TAT AGA AAC AAA GAT GAC AAA TGT GTA TC - #A GCA GAA GAC TGC GAA 243Phe Tyr Arg Asn Lys Asp Asp Lys Cys Val Se - #r Ala Glu Asp Cys Glu# 75- CTT GAC AAT ATG GAC TTT ATA TAT CCC GGA AC - #T CGA AAC TGAACGAAGG CTC 295Leu Asp Asn Met Asp Phe Ile Tyr Pro Gly Th - #r Arg Asn# 90- CATTCTTGCT GCACAAGATC GATTGTCTCT CCCCTGCATC TCAGTAGTTT TG - #CTACATTG 355- TATATGGTAG CAAAAAATTA GCTTAGGGAG AATAAAATCT TTACCTATAT TT - #AATCAATG 415# 430- (2) INFORMATION FOR SEQ ID NO: 48:- (i) SEQUENCE CHARACTERISTICS:# 100 amino acids:# amino acidE:# linear(D) TOPOLOGY:# 48: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Met Lys Met Leu Tyr Ala Ile Ala Ile Met Ph - #e Leu Leu Val Ser Leu# 15- Cys Ser Ala Arg Thr Val Arg Lys Ala Tyr Pr - #o Glu Cys Gly Glu Asn# 30- Glu Trp Leu Asp Asp Cys Gly Thr Gln Lys Pr - #o Cys Glu Ala Lys Cys# 45- Asn Glu Glu Pro Pro Glu Glu Glu Asp Pro Il - #e Cys Arg Ser Arg Gly# 60- Cys Leu Leu Pro Pro Ala Cys Val Cys Lys As - #p Gly Phe Tyr Arg Asp# 80- Thr Val Ile Gly Asp Cys Val Arg Glu Glu Gl - #u Cys Asp Gln His Glu# 95- Ile Ile His Val 100- (2) INFORMATION FOR SEQ ID NO: 49:- (i) SEQUENCE CHARACTERISTICS:# 98 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:# 49: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Met Lys Met Leu Tyr Ala Ile Ala Ile Met Ph - #e Leu Leu Val Ser Leu# 15- Cys Ser Thr Arg Thr Val Arg Lys Ala Tyr Pr - #o Glu Cys Gly Glu Asn# 30- Glu Trp Leu Asp Val Cys Gly Thr Lys Lys Pr - #o Cys Glu Ala Lys Cys# 45- Ser Glu Glu Glu Glu Glu Asp Pro Ile Cys Ar - #g Ser Phe Ser Cys Pro# 60- Gly Pro Ala Ala Cys Val Cys Glu Asp Gly Ph - #e Tyr Arg Asp Thr Val# 80- Ile Gly Asp Cys Val Lys Glu Glu Glu Cys As - #p Gln His Glu Ile Ile# 95- His Val- (2) INFORMATION FOR SEQ ID NO: 50:- (i) SEQUENCE CHARACTERISTICS:# 91 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:# 50: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Leu Val Ser Tyr Cys Ser Gly Lys Ala Thr Me - #t Gln Cys Gly Glu Asn# 15- Glu Lys Tyr Asp Ser Cys Gly Ser Lys Glu Cy - #s Asp Lys Lys Cys Lys# 30- Tyr Asp Gly Val Glu Glu Glu Asp Asp Glu Gl - #u Pro Asn Val Pro Cys# 45- Leu Val Arg Val Cys His Gln Asp Cys Val Cy - #s Glu Glu Gly Phe Tyr# 60- Arg Asn Lys Asp Asp Lys Cys Val Ser Ala Gl - #u Asp Cys Glu Leu Asp# 80- Asn Met Asp Phe Ile Tyr Pro Gly Thr Arg As - #n# 90- (2) INFORMATION FOR SEQ ID NO: 51:- (i) SEQUENCE CHARACTERISTICS:# 94 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:# 51: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Met Arg Thr Leu Tyr Leu Ile Ser Ile Trp Le - #u Phe Leu Ile Ser Gln# 15- Cys Asn Gly Lys Ala Phe Pro Lys Cys Asp Va - #l Asn Glu Arg Phe Glu# 30- Val Cys Gly Asn Leu Lys Glu Cys Glu Leu Ly - #s Cys Asp Glu Asp Pro# 45- Lys Ile Cys Ser Arg Ala Cys Ile Arg Pro Pr - #o Ala Cys Val Cys Asp# 60- Asp Gly Phe Tyr Arg Asp Lys Tyr Gly Phe Cy - #s Val Glu Glu Asp Glu# 80- Cys Asn Asp Met Glu Ile Ile Thr Phe Pro Pr - #o Glu Thr Lys# 90- (2) INFORMATION FOR SEQ ID NO: 52:- (i) SEQUENCE CHARACTERISTICS:# 96 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:# 52: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Met Ser Thr Leu Tyr Val Ile Ala Ile Cys Le - #u Leu Leu Val Ser Gln# 15- Cys Asn Gly Arg Thr Val Lys Lys Cys Gly Ly - #s Asn Glu Arg Tyr Asp# 30- Asp Cys Gly Asn Ala Lys Asp Cys Glu Thr Ly - #s Cys Gly Glu Glu Glu# 45- Lys Val Cys Arg Ser Arg Glu Cys Thr Ser Pr - #o Gly Ala Cys Val Cys# 60- Glu Gln Gly Phe Tyr Arg Asp Pro Ala Gly As - #p Cys Val Thr Asp Glu#80- Glu Cys Asp Glu Trp Asn Asn Met Glu Ile Il - #e Thr Met Pro Lys Gln# 95- (2) INFORMATION FOR SEQ ID NO: 53:- (i) SEQUENCE CHARACTERISTICS:# 108 amino acids:# amino acidE:# linear(D) TOPOLOGY:# 53: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Met Ala Val Leu Tyr Ser Val Ala Ile Ala Le - #u Leu Leu Val Ser Gln# 15- Cys Ser Gly Lys Pro Asn Asn Val Met Thr As - #n Ala Cys Gly Leu Asn# 30- Glu Tyr Phe Ala Glu Cys Gly Asn Met Lys Gl - #u Cys Glu His Arg Cys# 45- Asn Glu Glu Glu Asn Glu Glu Arg Asp Glu Gl - #u Arg Ile Thr Ala Cys# 60- Leu Ile Arg Val Cys Phe Arg Pro Gly Ala Cy - #s Val Cys Lys Asp Gly# 80- Phe Tyr Arg Asn Arg Thr Gly Ser Cys Val Gl - #u Glu Asp Asp Cys Glu# 95- Tyr Glu Asn Met Glu Phe Ile Thr Phe Ala Pr - #o Glu# 105- (2) INFORMATION FOR SEQ ID NO: 54:- (i) SEQUENCE CHARACTERISTICS:# 82 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:# 54: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Val Pro Ile Cys Gly Ser Asn Glu Arg Tyr Se - #r Asp Cys Gly Asn Asp# 15- Lys Gln Cys Glu Arg Lys Cys Asn Glu Asp As - #p Tyr Glu Lys Gly Asp# 30- Glu Ala Cys Arg Ser His Val Cys Glu Arg Pr - #o Gly Ala Cys Val Cys# 45- Glu Asp Gly Phe Tyr Arg Asn Lys Lys Gly Se - #r Cys Val Glu Ser Asp# 60- Asp Cys Glu Tyr Asp Asn Met Asp Phe Ile Th - #r Phe Ala Pro Glu Thr# 80- Ser Arg- (2) INFORMATION FOR SEQ ID NO: 55:- (i) SEQUENCE CHARACTERISTICS:# 76 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:# 55: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Lys Cys Pro Thr Asp Glu Trp Phe Asp Trp Cy - #s Gly Thr Tyr Lys His# 15- Cys Glu Leu Lys Cys Asp Arg Glu Leu Thr Gl - #u Lys Glu Glu Gln Ala# 30- Cys Leu Ser Arg Val Cys Glu Lys Ser Ala Cy - #s Val Cys Asn Asp Gly# 45- Leu Tyr Arg Asp Lys Phe Gly Asn Cys Val Gl - #u Lys Asp Glu Cys Asn# 60- Asp Met Glu Ile Ile Thr Phe Ala Pro Glu Th - #r Lys# 75- (2) INFORMATION FOR SEQ ID NO: 56:- (i) SEQUENCE CHARACTERISTICS:# 102 amino acids:# amino acidE:# linear(D) TOPOLOGY:# 56: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Met Arg Met Leu Tyr Leu Val Pro Ile Trp Le - #u Leu Leu Ile Ser Leu# 15- Cys Ser Gly Lys Ala Ala Lys Lys Cys Gly Le - #u Asn Glu Arg Leu Asp# 30- Cys Gly Asn Leu Lys Gln Cys Glu Pro Lys Cy - #s Ser Asp Leu Glu Ser# 45- Glu Glu Tyr Glu Glu Glu Asp Glu Ser Lys Cy - #s Arg Ser Arg Glu Cys# 60- Ser Arg Arg Val Cys Val Cys Asp Glu Gly Ph - #e Tyr Arg Asn Lys Lys# 80- Gly Lys Cys Val Ala Lys Asp Val Cys Glu As - #p Asp Asn Met Glu Ile# 95- Ile Thr Phe Pro Pro Glu 100- (2) INFORMATION FOR SEQ ID NO: 57:- (i) SEQUENCE CHARACTERISTICS:# 78 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:# 57: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Asp Glu Cys Gly Pro Asp Glu Trp Phe Asp Ty - #r Cys Gly Asn Tyr Lys# 15- Lys Cys Glu Arg Lys Cys Ser Glu Glu Thr Se - #r Glu Lys Asn Glu Glu# 30- Ala Cys Leu Ser Arg Ala Cys Thr Gly Arg Al - #a Cys Val Cys Lys Asp# 45- Gly Leu Tyr Arg Asp Asp Phe Gly Asn Cys Va - #l Pro His Asp Glu Cys# 60- Asn Asp Met Glu Ile Ile Thr Phe Pro Pro Gl - #u Thr Lys His#75- (2) INFORMATION FOR SEQ ID NO: 58:- (i) SEQUENCE CHARACTERISTICS:# 76 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:# 58: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Met Ile Arg Lys Leu Val Leu Leu Thr Ala Il - #e Val Thr Val Val Leu# 15- Ser Ala Lys Thr Cys Gly Pro Asn Glu Glu Ty - #r Thr Glu Cys Gly Thr# 30- Pro Cys Glu Pro Lys Cys Asn Glu Pro Met Pr - #o Asp Ile Cys Thr Leu# 45- Asn Cys Ile Val Asn Val Cys Gln Cys Lys Pr - #o Gly Phe Lys Arg Gly# 60- Pro Lys Gly Cys Val Ala Pro Gly Pro Gly Cy - #s Lys#75- (2) INFORMATION FOR SEQ ID NO: 59:- (i) SEQUENCE CHARACTERISTICS:# 187 base pairsH:# nucleic acid#single (C) STRANDEDNESS:# linear(D) TOPOLOGY:# 59: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- TTATTCGAAA CGATGTTCTC TCCAATTTTG TCCTTGGAAA TTATTTTAGC TA - #CTTTGCAA 60- TCTGTCTTCG CCCAGCCAGT TATCTCCACT ACCGTTGGTT CCGCTGCCGA GG - #GTTCTTTG 120- GACAAGAGGC CTATCCGCGG AATTCAGATC TGAATGCGGC CGCTCGAGAC TA - #GTGGATCC 180# 187- (2) INFORMATION FOR SEQ ID NO: 60:- (i) SEQUENCE CHARACTERISTICS:# 8 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2 to 8ION:#amino acid. is an#60: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5- (2) INFORMATION FOR SEQ ID NO: 61:- (i) SEQUENCE CHARACTERISTICS:# 7 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2 to 6ION:#amino acid. is an#61: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Xaa Xaa Xaa Xaa Cys1 5- (2) INFORMATION FOR SEQ ID NO: 62:- (i) SEQUENCE CHARACTERISTICS:# 6 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2 to 5ION:#amino acid. is an#62: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Xaa Xaa Xaa Cys 1 5- (2) INFORMATION FOR SEQ ID NO: 63:- (i) SEQUENCE CHARACTERISTICS:# 5 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2 to 4ION:#amino acid. is an#63: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Xaa Xaa Cys1 5- (2) INFORMATION FOR SEQ ID NO: 64:- (i) SEQUENCE CHARACTERISTICS:# 4 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2 to 3ION:#amino acid. is an#64: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Xaa Cys- (2) INFORMATION FOR SEQ ID NO: 65:- (i) SEQUENCE CHARACTERISTICS:# 21 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 1 to 3, or 5#is an amino acid.21#65: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa# 15- Xaa Xaa Xaa Xaa Xaa 20- (2) INFORMATION FOR SEQ ID NO: 66:- (i) SEQUENCE CHARACTERISTICS:# 20 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 1 to 3, or 5#is an amino acid.20#66: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa# 15- Xaa Xaa Xaa Xaa 20- (2) INFORMATION FOR SEQ ID NO: 67:- (i) SEQUENCE CHARACTERISTICS:# 19 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 1 to 3, or 5#is an amino acid.19#67: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa# 15- Xaa Xaa Xaa- (2) INFORMATION FOR SEQ ID NO: 68:- (i) SEQUENCE CHARACTERISTICS:# 18 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 1 to 3, or 5#is an amino acid.18#68: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa# 15- Xaa Xaa- (2) INFORMATION FOR SEQ ID NO: 69:- (i) SEQUENCE CHARACTERISTICS:# 17 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (ix) FEATURE:# Xaa in any location 1 to 3, or 5#is an amino acid.17- (v) FRAGMENT TYPE: internal fragment#69: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa# 15- Xaa- (2) INFORMATION FOR SEQ ID NO: 70:- (i) SEQUENCE CHARACTERISTICS:# 16 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 1 to 3, or 5#is an amino acid.16#70: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa# 15- (2) INFORMATION FOR SEQ ID NO: 71:- (i) SEQUENCE CHARACTERISTICS:# 15 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 1 to 3, or 5#is an amino acid.15#71: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa# 15- (2) INFORMATION FOR SEQ ID NO: 72:- (i) SEQUENCE CHARACTERISTICS:# 14 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 1 to 3, or 5#is an amino acid.14#72: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa# 10- (2) INFORMATION FOR SEQ ID NO: 73:- (i) SEQUENCE CHARACTERISTICS:# 13 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 1 to 3, or 5#is an amino acid.13#73: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa# 10- (2) INFORMATION FOR SEQ ID NO: 74:- (i) SEQUENCE CHARACTERISTICS:# 12 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 1 to 3, or 5#is an amino acid.12#74: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa# 10- (2) INFORMATION FOR SEQ ID NO: 75:- (i) SEQUENCE CHARACTERISTICS:# 11 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (ix) FEATURE:# Xaa in any location 1 to 3, or 5#is an amino acid.11- (v) FRAGMENT TYPE: internal fragment#75: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a# 10- (2) INFORMATION FOR SEQ ID NO: 76:- (i) SEQUENCE CHARACTERISTICS:# 10 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 1 to 3, or 5#is an amino acid.10#76: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa# 10- (2) INFORMATION FOR SEQ ID NO: 77:- (i) SEQUENCE CHARACTERISTICS:# 5 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2 to 5 is an amino aci - #d.#77: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Xaa Xaa Xaa 1 5- (2) INFORMATION FOR SEQ ID NO: 78:- (i) SEQUENCE CHARACTERISTICS:# 4 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2 to 4 is:#acid. an amino#78: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Xaa Xaa 1- (2) INFORMATION FOR SEQ ID NO: 79:- (i) SEQUENCE CHARACTERISTICS:# 6 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2 to 6 is:#acid. an amino#79: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Xaa Xaa Xaa Xaa 1 5- (2) INFORMATION FOR SEQ ID NO: 80:- (i) SEQUENCE CHARACTERISTICS:# 5 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2 to 5 is:#acid. an amino#80: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Xaa Xaa Xaa 1 5- (2) INFORMATION FOR SEQ ID NO: 81:- (i) SEQUENCE CHARACTERISTICS:# 4 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2 to 4 is:#acid. an amino#81: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Xaa Xaa 1- (2) INFORMATION FOR SEQ ID NO: 82:- (i) SEQUENCE CHARACTERISTICS:# 15 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2, orTION: 4 to 1 - #5 is an amino acid.#82: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa# 150- (2) INFORMATION FOR SEQ ID NO: 83:- (i) SEQUENCE CHARACTERISTICS:# 14 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2, orTION: 4 to 1 - #4 is an amino acid.#83: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa# 10- (2) INFORMATION FOR SEQ ID NO: 84:- (i) SEQUENCE CHARACTERISTICS:# 13 amino acidsH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2, orTION: 4 to 1 - #3 is an amino acid.#84: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa# 10- (2) INFORMATION FOR SEQ ID NO: 85:- (i) SEQUENCE CHARACTERISTICS:# 8 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2 to 7ION:#amino acid. is an#85: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5- (2) INFORMATION FOR SEQ ID NO: 86:- (i) SEQUENCE CHARACTERISTICS:# 7 amino acidsTH:# amino acidE:# linear(D) TOPOLOGY:- (v) FRAGMENT TYPE: internal fragment- (ix) FEATURE:# Xaa in any location 2 to 6ION:#amino acid. is an#86: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:- Cys Xaa Xaa Xaa Xaa Xaa Cys 1 5__________________________________________________________________________

Number	Name	Date
4458066	Caruthers et al.	Jul 1984
4683293	Craig	Jul 1987
4745051	Smith et al.	May 1988
4777242	Nelles	Oct 1988
4808537	Stroman et al.	Feb 1989
4812405	Lair et al.	Mar 1989
4818700	Cregg et al.	Apr 1989
4837148	Cregg	Jun 1989
4855231	Stroman et al.	Aug 1989
4857467	Sreekrishna et al.	Aug 1989
4879231	Stroman et al.	Nov 1989
4882279	Cregg	Nov 1989
4885242	Cregg	Dec 1989
4895800	Tschopp et al.	Jan 1990
5002876	Sreekrishma et al.	Mar 1991
5004688	Craig et al.	Apr 1991
5023236	Edgington et al.	Jun 1991
5032516	Cregg	Jul 1991
5106833	Broze et al.	Apr 1992
5122465	Cregg et al.	Jun 1992
5135868	Cregg	Aug 1992
5166329	Cregg	Nov 1992
5189019	Palladino et al.	Feb 1993
5204261	Prevatt et al.	Apr 1993
5239058	Vlasuk et al.	Aug 1993
5239059	Zasloff et al.	Aug 1993
5268273	Buckholz	Dec 1993
5330901	Prevatt et al.	Jul 1994
5427937	Cappello et al.	Jun 1995
5525477	Hassouna	Jun 1996
5605671	Lyle et al.	Feb 1997

Number	Date	Country
0255771	Feb 1988	EPX
0419099	Mar 1991	EPX
0439442	Jul 1991	EPX
0454372	Oct 1991	EPX
8809811	Dec 1988	WOX
9102753	Mar 1991	WOX
9425000	Nov 1994	WOX
9512615	May 1995	WOX
9604378	Feb 1996	WOX
9612021	Apr 1996	WOX
9604377	Feb 1997	WOX

Nematode-extracted anticoagulant protein

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US Referenced Citations (31)

Foreign Referenced Citations (11)

Non-Patent Literature Citations (102)

Entry
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