Streptococcus pneumoniae sequence GrpE

Abstract

The invention provides isolated nucleic acid compounds encoding GrpE of Streptococcus pneumoniae. Also provided are vectors and transformed host cells for expressing the encoded protein, and a method for identifying compounds that bind and/or inhibit said protein.

Description

BACKGROUND OF THE INVENTION

This invention provides isolated DNA sequences, proteins encoded thereby, and methods of using said DNA and protein in a variety of applications.

Widespread antibiotic resistance in common pathogenic bacterial species has justifiably alarmed the medical and research communities. Frequently, resistant organisms are co-resistant to several antibacterial agents. Penicillin resistance in Streptococcus pneumoniae has been particularly problematic. This organism causes upper respiratory tract infections. Modification of a penicillin-binding protein (PBP) underlies resistance to penicillin in the majority of cases. Combating resistance to antibiotic agents will require research into the molecular biology of pathogenic organisms. The goal of such research will be to identify new antibacterial agents.

While researchers continue to develop antibiotics effective against a number of microorganisms, Streptococcus pneumoniae has been more refractory. In part, this is because Streptococcus pneumoniae is highly recombinogenic and readily takes up exogenous DNA from its surroundings. Thus, there is a need for new antibacterial compounds and new targets for antibacterial therapy in Streptococcus pneumoniae.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to an isolated gene and encoded protein from S. pneumoniae. The invention enables: (1) preparation of probes and primers for use in hybridizations and PCR amplifications, (2) production of proteins and RNAs encoded by said gene and related nucleic acids, and (3) methods to identify compounds that bind and/or inhibit said protein(s).

In one embodiment the present invention relates to an isolated nucleic acid molecule encoding GrpE protein.

In another embodiment, the invention relates to a nucleic acid molecule comprising the nucleotide sequence identified as SEQ ID NO:1 or SEQ ID NO:3.

In another embodiment, the present invention relates to a nucleic acid that encodes SEQ ID NO:2.

In another embodiment the present invention relates to an isolated protein molecule, wherein said protein molecule comprises the sequence identified as SEQ ID NO:2.

In yet another embodiment, the present invention relates to a recombinant DNA vector that incorporates the GrpE gene in operable linkage to gene expression sequences enabling the gene to be transcribed and translated in a host cell.

In still another embodiment the present invention relates to host cells that have been transformed or transfected with the cloned GrpE gene such that said gene is expressed in the host cell.

This invention also provides a method of determining whether a nucleic acid sequence of the present invention, or fragment thereof, is present in a sample, comprising contacting the sample, under suitable hybridization conditions, with a nucleic acid probe of the present invention.

In a still further embodiment, the present invention relates to a method for identifying compounds that bind and/or inhibit the GrpE protein.

DETAILED DESCRIPTION OF THE INVENTION

“ORF” (i.e. “open reading frame”) designates a region of genomic DNA beginning with a Met or other initiation codon and terminating with a translation stop codon, that potentially encodes a protein product. “Partial ORF” means a portion of an ORF as disclosed herein such that the initiation codon, the stop codon, or both are not disclosed.

“Consensus sequence” refers to an amino acid or nucleotide sequence that may suggest the biological function of a protein, DNA, or RNA molecule. Consensus sequences are identified by comparing proteins, RNAs, and gene homologues from different species.

The terms “cleavage” or “restriction” of DNA refers to the catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA (viz. sequence-specific endonucleases). The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements are used in the manner well known to one of ordinary skill in the art. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer or can readily be found in the literature.

“Essential genes” or “essential ORFs” or “essential proteins” refer to genomic information or the protein(s) or RNAs encoded thereby, that when disrupted by knockout mutation, or by other mutation, result in a loss of viability of cells harboring said mutation.

“Non-essential genes” or “non-essential ORFs” or “non-essential proteins” refer to genomic information or the protein(s) or RNAs encoded therefrom which when disrupted by knockout mutation, or other mutation, do not result in a loss of viability of cells harboring said mutation.

“Minimal gene set” refers to a genus comprising about 256 genes conserved among different bacteria such as M. genitalium and H. influenzae. The minimal gene set may be necessary and sufficient to sustain life. See e.g. A. Mushegian and E. Koonin, “A minimal gene set for cellular life derived by comparison of complete bacterial genomes” Proc. Nat. Acad. Sci. 93, 10268-273 (1996).

“Knockout mutant” or “knockout mutation” as used herein refers to an in vitro engineered disruption of a region of native chromosomal DNA, typically within a protein coding region, such that a foreign piece of DNA is inserted within the native sequence. A knockout mutation occurring in a protein coding region prevents expression of the wild-type protein. This usually leads to loss of the function provided by the protein. A “knockout cassette” refers to a fragment of native chromosomal DNA having cloned therein a foreign piece of DNA that may provide a selectable marker.

The term “plasmid” refers to an extrachromosomal genetic element. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accordance with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

“Recombinant DNA cloning vector” as used herein refers to any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.

The term “recombinant DNA expression vector” as used herein refers to any recombinant DNA cloning vector, for example a plasmid or phage, in which a promoter and other regulatory elements are present to enable transcription of the inserted DNA.

The term “vector” as used herein refers to a nucleic acid compound used for introducing exogenous DNA into host cells. A vector comprises a nucleotide sequence which may encode one or more protein molecules. Plasmids, cosmids, viruses, and bacteriophages, in the natural state or which have undergone recombinant engineering, are examples of commonly used vectors.

The terms “complementary” or “complementarity” as used herein refer to the capacity of purine and pyrimidine nucleotides to associate through hydrogen bonding to form double stranded nucleic acid molecules. The following base pairs are related by complementarity: guanine and cytosine; adenine and thymine; and adenine and uracil. As used herein, “complementary” applies to all base pairs comprising two single-stranded nucleic acid molecules. “Partially complementary” means one of two single-stranded nucleic acid molecules is shorter than the other, such that one of the molecules remains partially single-stranded.

“Oligonucleotide” refers to a short nucleotide chain comprising from about 2 to about 25 nucleotides.

“Isolated nucleic acid compound” refers to any RNA or DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location.

A “primer” is a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation of, for example, a nucleic acid molecule.

The term “promoter” refers to a DNA sequence which directs transcription of DNA to RNA.

A “probe” as used herein is a labeled nucleic acid compound which can be used to hybridize with another nucleic acid compound.

The term “hybridization” or “hybridize” as used herein refers to the process by which a single-stranded nucleic acid molecule joins with a complementary strand through nucleotide base pairing.

“Substantially purified” as used herein means a specific isolated nucleic acid or protein, or fragment thereof, in which substantially all contaminants (i.e. substances that differ from said specific molecule) have been separated from said nucleic acid or protein. For example, a protein may, but not necessarily, be “substantially purified” by the IMAC method as described herein.

“Selective hybridization” refers to hybridization under conditions of high stringency. The degree of hybridization between nucleic acid molecules depends upon, for example, the degree of complementarity, the stringency of hybridization, and the length of hybridizing strands.

The term “stringency” relates to nucleic acid hybridization conditions. High stringency conditions disfavor non-homologous base pairing. Low stringency conditions have the opposite effect. Stringency may be altered, for example, by changes in temperature and salt concentration. Typical high stringency conditions comprise hybridizing at 50° C. to 65° C. in 5×SSPE and 50% formamide, and washing at 50° C. to 65° C. in 0.5×SSPE; typical low stringency conditions comprise hybridizing at 35° C. to 37° C. in 5×SSPE and 40% to 45% formamide and washing at 42° C. in 1×-2×SSPE.

“SSPE” denotes a hybridization and wash solution comprising sodium chloride, sodium phosphate, and EDTA, at pH 7.4. A 20×solution of SSPE is made by dissolving 174 g of NaCl, 27.6 g of NaH 2 PO4.H 2 O, and 7.4 g of EDTA in 800 ml of H 2 O. The pH is adjusted with NaOH and the volume brought to 1 liter.

“SSC” denotes a hybridization and wash solution comprising sodium chloride and sodium citrate at pH 7. A 20×solution of SSC is made by dissolving 175 g of NaCl and 88 g of sodium citrate in 800 ml of H 2 O. The volume is brought to 1 liter after adjusting the pH with ION NaOH.

The GrpE gene disclosed herein (SEQ ID NO:1) and related nucleic acids (e.g. SEQ ID NO:3 and SEQ ID NO:4) encode a heat shock protein that functions as a chaperone protein. GrpE is thought to stimulate the ATPase activity of DnaK.

The proteins categorized as “minimal gene set” counterparts are homologous to a set of highly conserved proteins found in other bacteria. The minimal gene set proteins are thought to be essential for viability and are useful targets for the development of new antibacterial compounds.

In one embodiment, the proteins of this invention are purified, and used in a screen to identify compounds that bind and/or inhibit the activity of said proteins. A variety of suitable screens are contemplated for this purpose. For example, the protein(s) can be labeled by known techniques, such as radiolabeling or fluorescent tagging, or by labeling with biotin/avidin. Thereafter, binding of a test compound to a labeled protein can be determined by any suitable means, well known to the skilled artisan.

Skilled artisans will recognize that the DNA molecules of this invention, or fragments thereof, can be generated by general cloning methods. PCR amplification using oligonucleotide primers targeted to any suitable region of SEQ ID NO:1 is preferred. Methods for PCR amplification are widely known in the art. See e.g. PCR Protocols: A Guide to Method and Application, Ed. M. Innis et al., Academic Press (1990) or U.S. Pat. No. 4,889,818, which hereby is incorporated by reference. A PCR comprises DNA, suitable enzymes, primers, and buffers, and is conveniently carried out in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, Conn.). A positive PCR result is determined by, for example, detecting an appropriately-sized DNA fragment following agarose gel electrophoresis.

The DNAs of the present invention may also be produced using synthetic methods well known in the art. (See, e.g., E. L. Brown, R. Belagaje, M. J. Ryan, and H. G. Khorana, Methods in Enzymology, 68:109-151 (1979)). An apparatus such as the Applied Biosystems Model 380A or 380B DNA synthesizers (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, Calif. 94404) may be used to synthesize DNA. Synthetic methods rely upon phosphotriester chemistry [See, e.g., M. J. Gait, ed., Oligonucleotide Synthesis, A Practical Approach, (1984)], or phosphoramidite chemistry.

Protein Production Methods

The present invention relates further to substantially purified proteins encoded by the gene disclosed herein.

Skilled artisans will recognize that proteins can be synthesized by different methods, for example, chemical methods or recombinant methods, as described in U.S. Pat. No. 4,617,149, which hereby is incorporated by reference.

The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts relating to this area. See, e.g., H. Dugas and C. Penney, Bioorganic Chemistry (1981) Springer-Verlag, New York, 54-92. Peptides may be synthesized by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, Calif.) and synthesis cycles supplied by Applied Biosystems. Protected amino acids, such as t-butoxycarbonyl-protected amino acids, and other reagents are commercially available from many chemical supply houses.

The proteins of the present invention can also be made by recombinant DNA methods. Recombinant methods are preferred if a high yield is desired. Recombinant methods involve expressing the cloned gene in a suitable host cell. The gene is introduced into the host cell by any suitable means, well known to those skilled in the art. While chromosomal integration of the cloned gene is within the scope of the present invention, it is preferred that the cloned gene be maintained extra-chromosomally, as part of a vector in which the gene is in operable-linkage to a promoter.

Recombinant methods can also be used to overproduce a membrane-bound or membrane-associated protein. In some cases, membranes prepared from recombinant cells expressing such proteins provide an enriched source of the protein.

Expressing Recombinant Proteins in Procaryotic and Eucaryotic Host Cells

Procaryotes are generally used for cloning DNA sequences and for constructing vectors. For example, the Escherichia coli K12 strain 294 (ATCC No. 31446) is particularly useful for expression of foreign proteins. Other strains of E. coli, bacilli such as Bacillus subtilis, enterobacteriaceae such as Salmonella typhimurium or Serratia marcescans, various Pseudomonas species may also be employed as host cells in cloning and expressing the recombinant proteins of this invention. Also contemplated are various strains of Streptococcus and Streptocmyces.

For effective recombinant protein production, a gene must be linked to a promoter sequence. Suitable bacterial promoters include β-lactamase [e.g. vector pGX2907, ATCC 39344, contains a replicon and β-lactamase gene], lactose systems [Chang et al., Nature (London), 275:615 (1978); Goeddel et al., Nature (London), 281:544 (1979)], alkaline phosphatase, and the tryptophan (trp) promoter system [vector pATH1 (ATCC 37695)] designed for the expression of a trpE fusion protein. Hybrid promoters such as the tac promoter (isolatable from plasmid pDR540, ATCC-37282) are also suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence, operably linked to the DNA encoding the desired polypeptides. These examples are illustrative rather than limiting.

A variety of mammalian cells and yeasts are also suitable hosts. The yeast Saccharomyces cerevisiae is commonly used. Other yeasts, such as Kluyveromyces lactis, are also suitable. For expression of recombinant genes in Saccharomyces, the plasmid YRp7 (ATCC-40053), for example, may be used. See, e.g., L. Stinchcomb, et al., Nature, 282:39 (1979); J. Kingsman et al., Gene, 7:141 (1979); S. Tschemper et al., Gene, 10:157 (1980). Plasmid YRp7 contains the TRP1 gene, a selectable marker for a trp1 mutant.

Purification of Recombinantly-Produced Protein

An expression vector carrying a nucleic acid or gene of the present invention is transformed or transfected into a suitable host cell using standard methods. Cells that contain the vector are propagated under conditions suitable for expression of a recombinant protein. For example, if the gene is under the control of an inducible promoter, then suitable growth conditions would incorporate the appropriate inducer. The recombinantly-produced protein may be purified from cellular extracts of transformed cells by any suitable means.

In a preferred process for protein purification a gene is modified at the 5′ end, or at some other position, such that the encoded protein incorporates several histidine residues (viz. “histidine tag”). This “histidine tag” enables “immobilized metal ion affinity chromatography” (IMAC), a single-step protein purification method described in U.S. Pat. No. 4,569,794, which hereby is incorporated by reference. The IMAC method enables isolation of substantially pure protein starting from a crude cellular extract.

As skilled artisans will recognize, owing to the degeneracy of the code, the proteins of the invention can be encoded by a large genus of different nucleic acid sequences. This invention further comprises said genus.

The ribonucleic acid compounds of the invention may be prepared using the polynucleotide synthetic methods discussed supra, or they may be prepared enzymatically using RNA polymerase to transcribe a DNA template.

The most preferred systems for preparing the ribonucleic acids of the present invention employ the RNA polymerase from the bacteriophage T7 or the bacteriophage SP6. These RNA polymerases are highly specific, requiring the insertion of bacteriophage-specific sequences at the 5′ end of a template. See, J. Sambrook, et al., supra, at 18.82-18.84.

This invention also provides nucleic acids that are complementary to the sequences disclosed herein.

The present invention also provides probes and primers, useful for a variety of molecular biology techniques including, for example, hybridization screens of genomic or subgenomic libraries, or detection and quantification of mRNA species as a means to analyze gene expression. A nucleic acid compound is provided comprising any of the sequences disclosed herein, or a complementary sequence thereof, or a fragment thereof, which is at least 15 base pairs in length, and which will hybridize selectively to Streptococcus pneumoniae DNA or mRNA. Preferably, the 15 or more base pair compound is DNA. A probe or primer length of at least 15 base pairs is dictated by theoretical and practical considerations. See e.g. B. Wallace and G. Miyada, “Oligonucleotide Probes for the Screening of Recombinant DNA Libraries,” In Methods in Enzymology, Vol. 152, 432-442, Academic Press (1987).

The probes and primers of this invention can be prepared by methods well known to those skilled in the art (See e.g. Sambrook et al. supra). In a preferred embodiment the probes and primers are synthesized by the polymerase chain reaction (PCR).

The present invention also relates to recombinant DNA cloning vectors and expression vectors comprising the nucleic acids of the present invention. Preferred nucleic acid vectors are those that comprise DNA. The skilled artisan understands that choosing the most appropriate cloning vector or expression vector depends on the availability of restriction sites, the type of host cell into which the vector is to be transfected or transformed, the purpose of transfection or transformation (e.g., stable transformation as an extrachromosomal element, or integration into a host chromosome), the presence or absence of readily assayable or selectable markers (e.g., antibiotic resistance and metabolic markers of one type and another), and the number of gene copies desired in the host cell.

Suitable vectors comprise RNA viruses, DNA viruses, lytic bacteriophages, lysogenic bacteriophages, stable bacteriophages, plasmids, viroids, and the like. The most preferred vectors are plasmids.

Host cells harboring the nucleic acids disclosed herein are also provided by the present invention. A preferred host is E. coli transfected or transformed with a vector comprising a nucleic acid of the present invention.

The invention also provides a host cell capable of expressing a gene described herein, said method comprising transforming or otherwise introducing into a host cell a recombinant DNA vector comprising an isolated DNA sequence that encodes said gene. The preferred host cell is any strain of E. coli that can accommodate high level expression of an exogenously introduced gene. Transformed host cells are cultured under conditions well known to skilled artisans, such that said gene is expressed, thereby producing the encoded protein in the recombinant host cell.

To discover compounds having antibacterial activity, one can look for agents that inhibit cell growth and/or viability by, for example, inhibiting enzymes required for cell wall biosynthesis, and/or by identifying agents that interact with membrane proteins. A method for identifying such compounds comprises contacting a suitable protein or membrane preparation with a test compound and monitoring by any suitable means an interaction and/or inhibition of a protein of this invention.

For example, the instant invention provides a screen for compounds that interact with the proteins of the invention, said screen comprising:

a) preparing a protein, or membranes enriched in a protein;

b) exposing the protein or membranes to a test compound; and

c) detecting an interaction of a protein with said compound by any suitable means.

The screening method of this invention may be adapted to automated procedures such as a PANDEX® (Baxter-Dade Diagnostics) system, allowing for efficient high-volume screening of compounds.

In a typical screen, a protein is prepared as described herein, preferably using recombinant DNA technology. A test compound is introduced into a reaction vessel containing said protein. The reaction/interaction of said protein and said compound is monitored by any suitable means. In a preferred method, a radioactively-labeled or chemically-labeled compound or protein is used. A specific association between the test compound and protein is monitored by any suitable means.

In such a screening protocol GrpE is prepared as described herein, preferably using recombinant DNA technology. A test compound is introduced into a reaction vessel containing the GrpE protein or fragment thereof. Binding of GrpE by a test compound is determined by any suitable means. For example, in one method radioactively-labeled or chemically-labeled test compound may be used. Binding of the protein by the compound is assessed, for example, by quantifying bound label versus unbound label using any suitable method. Binding of a test compound may also be carried out by a method disclosed in U.S. Pat. No. 5,585,277, which hereby is incorporated by reference. In this method, binding of a test compound to a protein is assessed by monitoring the ratio of folded protein to unfolded protein, for example by monitoring sensitivity of said protein to a protease, or amenability to binding of said protein by a specific antibody against the folded state of the protein.

The foregoing screening methods are useful for identifying a ligand of a GrpE protein, perhaps as a lead to a pharmaceutical compound for modulating the state of differentiation of an appropriate tissue. A ligand that binds GrpE, or related fragment thereof, is identified, for example, by combining a test ligand with GrpE under conditions that cause the protein to exist in a ratio of folded to unfolded states. If the test ligand binds the folded state of the protein, the relative amount of folded protein will be higher than in the case of a test ligand that does not bind the protein. The ratio of protein in the folded versus unfolded state is easily determinable by, for example, susceptibility to digestion by a protease, or binding to a specific antibody, or binding to chaperonin protein, or binding to any suitable surface.

The following examples more fully describe the present invention. Those skilled in the art will recognize that the particular reagents, equipment, and procedures described are merely illustrative and are not intended to limit the present invention in any manner.

EXAMPLE 1

Production of a Vector for Expressing S. pneumoniae GrpE in a Host Cell

An expression vector suitable for expressing S. pneumoniae GrpE in a variety of procaryotic host cells, such as E. coli, is easily made. The vector contains an origin of replication (Ori), an ampicillin resistance gene (Amp) useful for selecting cells which have incorporated the vector following a tranformation procedure, and further comprises the T7 promoter and T7 terminator sequences in operable linkage to the GrpE coding region. Plasmid pET11A (obtained from Novogen, Madison, Wis.) is a suitable parent plasmid. pET11A is linearized by restriction with endonucleases NdeI and BamHI. Linearized pET11A is ligated to a DNA fragment bearing NdeI and BamHI sticky ends and comprising the coding region of the S. pneumoniae GrpE (SEQ ID NO:1). The coding region for GrpE is easily produced by PCR technology using suitably designed primers to the ends of the coding region specified in SEQ ID NO:1.

The GrpE encoding nucleic acid used in this construct is slightly modified at the 5′ end (amino terminus of encoded protein) in order to simplify purification of the encoded protein product. For this purpose, an oligonucleotide encoding 8 histidine residues is inserted after the ATG start codon. Placement of the histidine residues at the amino terminus of the encoded protein serves to enable the IMAC one-step protein purification procedure.

EXAMPLE 2

Recombinant Expression and Purification of a Protein Encoded by S. pneumoniae GrpE

An expression vector that carries GrpE from the S. pneumoniae genome as disclosed herein and which GrpE is operably-linked to an expression promoter is transformed into E. coli BL21 (DE3) (hsdS gal lcIts857 ind1Sam7nin5lacUV5-T7gene 1) using standard methods (see Example 4). Transformants, selected for resistance to ampicillin, are chosen at random and tested for the presence of the vector by agarose gel electrophoresis using quick plasmid preparations. Colonies which contain the vector are grown in L broth and the protein product encoded by the vector-borne ORF is purified by immobilized metal ion affinity chromatography (IMAC), essentially as described in U.S. Pat. No. 4,569,794.

Briefly, the IMAC column is prepared as follows. A metal-free chelating resin (e.g. Sepharose 6B IDA, Pharmacia) is washed in distilled water to remove preservative substances and infused with a suitable metal ion [e.g. Ni(II), Co(II), or Cu(II)] by adding a 50 mM metal chloride or metal sulfate aqueous solution until about 75% of the interstitial spaces of the resin are saturated with colored metal ion. The column is then ready to receive a crude cellular extract containing the recombinant protein product.

After removing unbound proteins and other materials by washing the column with any suitable buffer, pH 7.5, the bound protein is eluted in any suitable buffer at pH 4.3, or preferably with an imidizole-containing buffer at pH 7.5.

4 522 base pairs nucleic acid single linear DNA (genomic) NO NO CDS 1..522 1ATG GCC CAA GAT ATA AAA AAT GAA GAA GTA GAA GAA GTT CAA GAA GAG 48Met Ala Gln Asp Ile Lys Asn Glu Glu Val Glu Glu Val Gln Glu Glu 1 5 10 15GAA GTT GTG GAA ACA GCT GAA GAA ACA ACT CCT GAA AAG TCT GAG TTG 96Glu Val Val Glu Thr Ala Glu Glu Thr Thr Pro Glu Lys Ser Glu Leu 20 25 30GAC TTG GCA AAT GAA CGT GCA GAT GAG TTC GAA AAC AAA TAT CTT CGC 144Asp Leu Ala Asn Glu Arg Ala Asp Glu Phe Glu Asn Lys Tyr Leu Arg 35 40 45GCT CAT GCA GAA ATG CAA AAT ATC CAA CGC CGT GCC AAT GAA GAA CGT 192Ala His Ala Glu Met Gln Asn Ile Gln Arg Arg Ala Asn Glu Glu Arg 50 55 60CAA AAC TTG CAA CGT TAT CGT AGC CAG GAC TTG GCA AAA GCA ATC TTA 240Gln Asn Leu Gln Arg Tyr Arg Ser Gln Asp Leu Ala Lys Ala Ile Leu 65 70 75 80CCA TCT CTT GAC AAC CTT GAG CGT GCA CTT GCA GTT GAA GGT TTG ACA 288Pro Ser Leu Asp Asn Leu Glu Arg Ala Leu Ala Val Glu Gly Leu Thr 85 90 95GAT GAT GTG AAG AAG GGC TTG GCG ATG GTG CAA GAA AGC TTG ATT CAC 336Asp Asp Val Lys Lys Gly Leu Ala Met Val Gln Glu Ser Leu Ile His 100 105 110GCT TTG AAA GAA GAA GGA ATT GAA GAA ATC GCA GCA GAT GGC GAA TTT 384Ala Leu Lys Glu Glu Gly Ile Glu Glu Ile Ala Ala Asp Gly Glu Phe 115 120 125GAC CAT AAC TAC CAT ATG GCC ATC CAA ACT CTC CCA GGA GAC GAT GAA 432Asp His Asn Tyr His Met Ala Ile Gln Thr Leu Pro Gly Asp Asp Glu 130 135 140CAC CCA GTA GAT ACC ATC GCC CAA GTC TTT CAA AAA GGC TAC AAA CTC 480His Pro Val Asp Thr Ile Ala Gln Val Phe Gln Lys Gly Tyr Lys Leu145 150 155 160CAT GAC CGC ATC CTA CGC CCA GCA ATG GTA GTG GTG TAT AAC 522His Asp Arg Ile Leu Arg Pro Ala Met Val Val Val Tyr Asn 165 170 174 amino acids amino acid linear protein 2Met Ala Gln Asp Ile Lys Asn Glu Glu Val Glu Glu Val Gln Glu Glu 1 5 10 15Glu Val Val Glu Thr Ala Glu Glu Thr Thr Pro Glu Lys Ser Glu Leu 20 25 30Asp Leu Ala Asn Glu Arg Ala Asp Glu Phe Glu Asn Lys Tyr Leu Arg 35 40 45Ala His Ala Glu Met Gln Asn Ile Gln Arg Arg Ala Asn Glu Glu Arg 50 55 60Gln Asn Leu Gln Arg Tyr Arg Ser Gln Asp Leu Ala Lys Ala Ile Leu 65 70 75 80Pro Ser Leu Asp Asn Leu Glu Arg Ala Leu Ala Val Glu Gly Leu Thr 85 90 95Asp Asp Val Lys Lys Gly Leu Ala Met Val Gln Glu Ser Leu Ile His 100 105 110Ala Leu Lys Glu Glu Gly Ile Glu Glu Ile Ala Ala Asp Gly Glu Phe 115 120 125Asp His Asn Tyr His Met Ala Ile Gln Thr Leu Pro Gly Asp Asp Glu 130 135 140His Pro Val Asp Thr Ile Ala Gln Val Phe Gln Lys Gly Tyr Lys Leu145 150 155 160His Asp Arg Ile Leu Arg Pro Ala Met Val Val Val Tyr Asn 165 170 522 base pairs nucleic acid single linear mRNA NO NO 3AUGGCCCAAG AUAUAAAAAA UGAAGAAGUA GAAGAAGUUC AAGAAGAGGA AGUUGUGGAA 60ACAGCUGAAG AAACAACUCC UGAAAAGUCU GAGUUGGACU UGGCAAAUGA ACGUGCAGAU 120GAGUUCGAAA ACAAAUAUCU UCGCGCUCAU GCAGAAAUGC AAAAUAUCCA ACGCCGUGCC 180AAUGAAGAAC GUCAAAACUU GCAACGUUAU CGUAGCCAGG ACUUGGCAAA AGCAAUCUUA 240CCAUCUCUUG ACAACCUUGA GCGUGCACUU GCAGUUGAAG GUUUGACAGA UGAUGUGAAG 300AAGGGCUUGG CGAUGGUGCA AGAAAGCUUG AUUCACGCUU UGAAAGAAGA AGGAAUUGAA 360GAAAUCGCAG CAGAUGGCGA AUUUGACCAU AACUACCAUA UGGCCAUCCA AACUCUCCCA 420GGAGACGAUG AACACCCAGU AGAUACCAUC GCCCAAGUCU UUCAAAAAGG CUACAAACUC 480CAUGACCGCA UCCUACGCCC AGCAAUGGUA GUGGUGUAUA AC 522 1229 base pairs nucleic acid single linear DNA (genomic) NO NO 4ACAACGGATA ATGTCATCGA TCTCTTTGAA CACATCTTTA AGGAATGTTC AACGAAAACA 60TTGTGATGGC GGGCAAGGTC AATCTCTTGA ATTTTGCCAA TCTAGCAGCC TATCAGTTCT 120TTGACCAACC GCAAAAGGTG GCCTTGGAGA TTCGTGAGGG GTTGCGTGAG GATCAGATGC 180AAAATGTTCG TGTTGCAGAC GGTCAAGAGT CCTGTTTAGC TGACCTAGCG GTGATTAGTA 240GTAAGTTCCT CATTCCTTAT CGGGGAGTTG GAATTCTAGC CATTATCGGT CCAGTTAATC 300TGGATTACCA ACAGCTAATC AATCAAATCA ATGTGGTCAA CCGTGTTTTG ACCATGAAGT 360TGACAGATTT TTACCGCTAC CTCAGCAGTA ATCATTACGA AGTACATTAA GATTGAAATC 420ATTAAAGGAG GCGAACATGG CCCAAGATAT AAAAAATGAA GAAGTAGAAG AAGTTCAAGA 480AGAGGAAGTT GTGGAAACAG CTGAAGAAAC AACTCCTGAA AAGTCTGAGT TGGACTTGGC 540AAATGAACGT GCAGATGAGT TCGAAAACAA ATATCTTCGC GCTCATGCAG AAATGCAAAA 600TATCCAACGC CGTGCCAATG AAGAACGTCA AAACTTGCAA CGTTATCGTA GCCAGGACTT 660GGCAAAAGCA ATCTTACCAT CTCTTGACAA CCTTGAGCGT GCACTTGCAG TTGAAGGTTT 720GACAGATGAT GTGAAGAAGG GCTTGGCGAT GGTGCAAGAA AGCTTGATTC ACGCTTTGAA 780AGAAGAAGGA ATTGAAGAAA TCGCAGCAGA TGGCGAATTT GACCATAACT ACCATATGGC 840CATCCAAACT CTCCCAGGAG ACGATGAACA CCCAGTAGAT ACCATCGCCC AAGTCTTTCA 900AAAAGGCTAC AAACTCCATG ACCGCATCCT ACGCCCAGCA ATGGTAGTGG TGTATAACTA 960AGATACAAAC GCTCGTAAAA AGCTCGCAGT AAAAATAGGA GATTGACGAG TGTTCGATGA 1020ACACAAGAAA ATCTATCTTT TTTACTCAGA GCTTAGGGCG TGTTCGATTC GGCAATTCTG 1080ACGGTAGCTA AAGCAACTCG TCAGAAAACG GCAATCGCTA TGACGTTTGC CTAGCTTCCT 1140TACTAACTCG TCGTCGAAAT AAAATCGATT TCGACTCCTC GTGTCGCAAT TTACATAATA 1200GAAAACTTGT CCGAACGACA TAAACTATG 1229

Claims

1. An isolated protein comprising the amino acid sequence shown in SEQ ID NO:2.

2. An isolated protein consisting of the amino acid sequence shown in SEQ ID NO:2.