Preparation of a recombinant protein in a prokaryotic host cell

The instant application claims the benefit of provisional U.S. Appl. No. 60/229,010, filed Aug. 30, 2000, which is herein incorporated by reference and DE 100 37 111.6, filed July 27, 2000 (in the German language), which is herein incorporated by reference.

The present invention relates to advantageous processes for preparing heterologous proteins in prokaryotic host cells by improved codon use and/or expression of tRNAs which code for rarely occurring codons in the said host cell. Numerous bacteria, particularly Gram-positive bacteria, for example, have already been used as host cells for the preparation of heterologous recombinant proteins, e.g. secreted proteins, in an endotoxin-free environment (references 8, 9, 19). The bacterium Staphylococcus carnosus ( S. carnosus ), for example, which is used in the food industry for the fermentation of meat and fish, is a suitable bacterium for this purpose. It is free from virulence factors and proteolytic activities in the supernatant and is capable of secreting large amounts of protein (10). Moreover, only small amounts of host cell-coded proteins are secreted, which makes it easier to purify the protein produced. Bacterial enzymes (7, 4, 17), for example, have already been recombinantly produced and expressed in S. carnosus . Recombinant S. carnosus strains which express antigens such as the Streptococcus protein G on their surface,are particularly promising as live vaccines (5, 12).

One crucial disadvantage of numerous bacterial systems is their use of rare codons, which is very different from the codon preference in human genes. The presence of rare codons in E. coli led to delayed and reduced expression of recombinant genes (2, 6). The problem of the present invention is therefore to overcome this disadvantage of the prior art and provide a prokaryotic expression system with improved properties.

The problem is solved within the scope of the claims and specification of the present invention.

The use of the singular or plural in the claims or specification is not intended to be limiting in any way and also includes the alternative form.

The invention relates to a process for preparing a heterologous recombinant protein in a prokaryotic host cell, characterized in that the codon use of the host cell for host cell genes is determined, in the nucleic acid coding for the heterologous recombinant protein in the host cell rarely occurring codons are replaced by frequent codons, said host cell is transformed with said nucleic acid coding for the recombinant protein and said recombinant nucleic acid is expressed. With a process according to the invention it is possible to achieve a significantly better expression rate of the heterologous proteins compared with the process known from the prior art. The processes according to the invention are also particularly advantageous for the expression of recombinant proteins on the surface of prokaryotes, as the covalent anchoring on the cell wall depends on intact C-termini, which are absent from the secreted truncated proteins which are not prepared by the process according to the invention.

By heterologous is meant that said protein in said prokaryotic host cell is not native, i.e. it occurs as a protein peculiar to the host cell. “Recombinant” means produced by molecular-biological methods. A heterologous recombinant protein may be any protein known to the skilled person, such as, for example, insulin, hGH, tPA, cytokines, such as e.g. interleukins (IL) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega or IFN tau, tumour necrosis factor (TNF), TNF alpha and TNF beta, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF. Prokaryotic host cells may be any host cells known to the skilled person, particularly Gram-positive and Gram-negative host cells. These may be, for example, Escherichia coli, Bacillus subtilis , Streptomyces, Proteus mirabilis or preferably Staphylococcus, such as e.g. Staphylococcus carnosus in particular, as available from public collections, e.g. the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, e.g. Strain TM300 (DSM 4600, 4601 or 4602).

Within the scope of the process according to the invention, first of all the use of the codons, i.e. the coding nucleotide triplets, is analysed for said host cell for as many native proteins as possible in the basic coding gene sequence. The codon frequencies of 51 genes were analysed for the host organism Staphylococcus carnosus Strain TM300 (10) by way of example and are shown in the following Table 1:

It was found that coding sequences of Staphylococci for example have a very low G+C content of about 30% and the A+T-rich codons are preferred. Therefore according to the invention the production of recombinant proteins, for example hGH, is increased in two ways: (i) exchanging rare codons in the coding gene sequence for frequent ones (cf. also Example 1) and/or (ii) expressing rare tRNAs (tRNA=transfer RNA; cf. also Example 2).

The invention therefore also relates to processes for preparing a heterologous recombinant protein, wherein for S. carnosus , as shown in Table 1, or in similar manner for every other host cell according to the invention, either some or all of the rarely occurring codons are replaced by frequent ones, for example according to the analysis in Table 1 the Glu codon GAG is replaced by GAA or the Leu-codons CTC, CTG and CTA are replaced by TTA, TTG or CTT.

The invention therefore further relates to a process for preparing a heterologous recombinant protein in a prokaryotic host cell, characterised in that the codon use of the host cell for host cell genes is determined, said host cell is transformed with the nucleic acid which codes tRNA or tRNAs specific for rarely occurring codons and with said nucleic acid coding for the recombinant protein, and said recombinant nucleic acid is expressed (cf. also Example 2). The skilled person can determine the rare codons of the host cell by analyzing the genes or the cDNAs (or by reverse transcription of the mRNA). This has been done, for example, for the host cell S. carnosus Strain TM300 (10) in Table 1. The invention includes the introduction of tRNAs or nucleic acid coding therefore, for one to all the rare tRNAs in the host cell. Because the host cell is equipped with tRNAs which are specific for rare codons, the heterologous recombinant protein according to the invention is expressed in larger amounts than is the case in known processes from the prior art. This surprisingly advantageous property of the process according to the invention is also shown in Example 2. This may occur either on its own or in conjunction with the nucleic acid coding for the heterologous protein.

Therefore the invention includes a preferred process according to the invention which is characterized in that, in the nucleic acid coding for the heterologous recombinant protein in the host cell, rarely occurring codons are replaced by frequent codons and said host cell is transformed with tRNA or tRNAs coding for rarely occurring codons and with said nucleic acid coding for the recombinant protein.

The invention includes a preferred process according to the invention which is characterized in that codons which are used in the host cell with an average frequency of less than 15% are replaced by codons with an average frequency of at least 15%; preferably characterized in that codons which are used in the host cell with an average frequency of 7 to 12% are replaced by codons with an average frequency of more than 12%; most preferably characterized in that codons which are used in the host cell with an average frequency of up to 7 or 10% are replaced by codons with an average frequency of more than 7% or more than 10%. Another preferred embodiment is a preferred process according to the invention which is characterized in that the codon or codons which occur least often in said host cell is replaced by the codon or codons which occur most frequently in said host cell. One exception may be if said codons are absolutely essential for the expression.

The invention further includes a preferred process according to the invention which is characterized in that in the coding nucleic acid codons which are used in the host cell with an average frequency of less than 10% are replaced by codons with an average frequency of at least 10%.

The invention further includes a preferred process according to the invention which is characterized in that the host cell is Escherichia coli , as available from public collections, e.g. the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, e.g. E. coli Strain K12 JM107 (DSM 3950).

The invention further includes a preferred process according to the invention which is characterized in that the host cell is a host cell selected from the genus Staphylococcus.

The invention further includes a preferred process according to the invention which is characterized in that the host cell is Staphylococcus carnosus (cf. also Examples 1 and 2).

In a preferred process according to the invention the recombinant protein is an antibody protein. By antibody protein is also meant a fragment, e.g. a Fab fragment (in English “Fragment antigen-binding=Fab”), a F(ab′) 2 fragment, an Fv fragment (in English: “fragment variable”=fragment of the variable part) or a single-chain Fv (scFv)), minibodies, dia-, tria- and tetrabodies.

In a preferred process according to the invention the recombinant protein is insulin.

In a preferred process according to the invention the recombinant protein is tissue plasminogen activator (tPa).

In a preferred process according to the invention the recombinant protein is a human protein.

In a preferred process according to the invention the recombinant protein is growth hormone.

In a particularly preferred process according to the invention the recombinant protein is human growth hormone (hGH, cf. Examples 1 and 2).

Another preferred process according to the invention is characterized in that G- or C-rich codons are replaced by codons containing A or T. This means that codons which contain more G or C are replaced by codons which now contain, for example, an A or a T instead of a G or C; it does not mean that the codons must now contain exclusively G or C (cf. the most preferred embodiments hereinafter).

In the process according to the invention all the rare codons found may be replaced by frequent ones (cf. e.g. Table 1) and not only those specified in the preferred embodiments given hereinafter.

Another particularly preferred process according to the invention is characterized in that the codon GCC is replaced by GCA.

Another particularly preferred process according to the invention is characterized in that the codon TGC is replaced by TGT.

Another particularly preferred process according to the invention is characterized in that the codon GAC is replaced by GAT.

Another particularly preferred process according to the invention is characterized in that the codon GAG is replaced by GAA.

Another particularly preferred process according to the invention is characterized in that the codon TTC is replaced by TTT.

Another particularly preferred process according to the invention is characterized in that the codon GGG is replaced by GGT.

Another particularly preferred process according to the invention is characterized in that the codon CAC is replaced by CAT.

Another particularly preferred process according to the invention is characterized in that the codon ATA is replaced by ATT.

Another particularly preferred process according to the invention is characterized in that the codon AAG is replaced by AAA.

Another particularly preferred process according to the invention is characterized in that the codon CTC is replaced by TTA.

Another particularly preferred process according to the invention is characterized in that the codon AAC is replaced by AAT.

Another particularly preferred process according to the invention is characterized in that the codon CCC is replaced by CCA.

Another particularly preferred process according to the invention is characterized in that the codon CAG is replaced by CAA.

Another particularly preferred process according to the invention is characterized in that the codon CGG is replaced by CGT.

Another particularly preferred process according to the invention is characterized in that the codon TCG is replaced by TCA.

Another particularly preferred process according to the invention is characterized in that the codon ACC is replaced by ACA.

Another particularly preferred process according to the invention is characterized in that the codon GTC is replaced by GTT.

Another particularly preferred process according to the invention is characterized in that the codon TAC is replaced by TAT.

Another particularly preferred process according to the invention is characterized in that the codon TGA is replaced by TAA.

Yet another preferred process according to the invention is characterized in that the host cell is transformed with the nucleic acid coding for AGG-tRNA or AGA-tRNA and with said nucleic acid coding for the recombinant protein.

The invention also particularly includes a nucleic acid molecule coding for a recombinant heterologous protein, characterized in that at least one codon rarely occurring in the host cell has been replaced by a codon which occurs frequently in the host cell. By way of example the codons for the host cell S. carnosus were analysed in Table 1. The invention includes nucleic acid molecules wherein for S. carnosus or similarly for every other host cell according to the invention either some or all of the rarely occurring codons are replaced by frequent ones, for example the Glu codon GAG is replaced by GAA or the Leu codons CTC, CTG and CTA are replaced by TIA, TTG or CTT.

The invention includes a nucleic acid molecule coding for a recombinant heterologous protein, characterized in that codons which are used in the host cell with an average frequency of less than 15% are replaced by codons with an average frequency of at least 15%; preferably characterized in that codons which are used in the host cell with an average frequency of 7 to 12% are replaced by codons with an average frequency of more than 12%; most preferably characterized in that codons which are used in the host cell with an average frequency of up to 7 or 10% are replaced by codons with an average frequency of more than 7% or more than 10%. Another preferred embodiment is a nucleic acid molecule coding for a recombinant heterologous protein, characterized in that the codon or codons which occur least often in said host cell is or are replaced by the codon or codons occurring most frequently in said host cell. One exception may be if said codons are absolutely necessary for the expression.

The invention therefore relates in a preferred embodiment to a nucleic acid molecule, characterized in that codons which are used in the host cell with an average frequency of less than 10% are replaced by codons with an average frequency of at least 10%.

The invention further relates to a nucleic acid molecule according to the invention, characterized in that it codes for the human growth hormone hGH.

The invention relates to a nucleic acid molecule, characterized in that it comprises a nucleotide sequence from the following group: the following sequence

(SEQ:ID-NO.1) or a partial sequence thereof, a nucleic acid which can hybridise with said sequence under stringent conditions, an allelic variant or a functional variant of said sequence or a variant of the nucleic acid on the basis of the degenerative code. N within the scope of the invention denotes any nucleotide known to the skilled person.

The invention further relates to a nucleic acid molecule coded by the nucleotide sequence of SEQ:ID-NO.1

The abovementioned nucleic acid molecule according to the invention codes for hGH with the amino acid sequence of SEQ:ID-NO.2

according to the invention.

The invention relates to a vector containing a nucleic acid molecule according to the invention.

Another important embodiment of the invention is a host cell containing a nucleic acid molecule according to the invention or a vector according to the invention.

Another important embodiment of the invention is a host cell containing one or more tRNA molecules which codes for codons occurring rarely in this host cell.

A host cell of the genus Staphylococcus is preferred, while Staphylococcus carnosus , e.g. Strain TM300 (DSM 4600, 4601 or 4602) is most preferred.

The following Examples are intended to make the invention easier to understand and should in no way be regarded as limiting the scope of the invention.

Construction of a Synthetic hgH Gene with S. carnosus -specific Condon Use

As an example of a protein, human growth hormone (hGH) was expressed in S. carnosus Strain TM300.

Comparison of the codon use of the mature hGH with that shown in S. carnosus showed that 51 of the codons of the mature hGH, which represent 27% of the total number of codons, make up only 7% or less of the S. carnosus codon frequency (Tab. 2). An enterokinase cutting site was introduced at the 5′ end of the DNA sequence of the mature hGH and the hGH was synthesised with S. carnosus -specific codon use. The codons in the synthetic gene were used in about the same frequency as in the S. carnosus gene. Codons which were used by S. carnosus in a frequency of less than 10% were not used, apart from those which were needed to introduce restriction cutting sites (cf. Tab. 2).

The synthetic hGH DNA sequence was synthesised from overlapping oligonucleotides which were hybridised and ligated in vitro. The resulting DNA fragment was cloned into the E. coli plasmid pSL1190 (3), verified by sequencing and inserted in the hGH expression cassette in order to replace the hGH cDNA between the NsiI and PstI cutting sites (FIG. 1 ). The new expression cassette was isolated as a BglII-ClaI fragment, cloned into the compatible BamHI and NarI cutting sites of the Staphylococci-expression vector pTX15 and transformed in S. carnosus TM300 by protoplast transformation. In the expression vector obtained, the lip-hGH fusion gene was expressed under the control of the xyl promoter of pTX15. The expression was obtained, after the inactivation of XylR, the repressor of the xyl promoter, by the addition of 0.5% xylose to the nutrient medium. The in vitro cloning and sequencing were carried out by standard methods (1). The expression of the hGH gene was performed as a fusion with the lip-signal and propeptide from Staphylococcus hyicus , which contains an enterokinase cutting site which allows the release of the mature hGH with the correct N-terminus from the fusion protein (FIG. 1 ). The resulting expression vector pTX-LipP-hGH4 is identical to the vector pTX-LipP-hGH2 apart from the codon use of the mature hGH. The S. carnosus strains containing one of the plasmids were cultivated in a modified LB medium (1% soya bean extract, 0.5% yeast extract, 0.5% NaCl) supplemented with 0.5% xylose, to activate the xyl promoter of the expression vectors. The use of the improved hGH DNA had a profound effect on the efficiency of the production of hGH in S. carnosus . The Lip-hGH yield was improved at least twofold and the amount of shortened Lip-hGH protein was significantly reduced (FIG. 2, traces 2 and 3). The shortened Lip-hGH proteins are not the result of extracellular proteolytic activities, as the Lip-hGH protein is stable in the S. carnosus supernatant. The reduced presence of the shortened proteins in the supernatant of S. carnosus (pTX-LipP-hGH4) shows that hGH is translated faster by the optimised DNA sequence and thus gives intracellular proteases less opportunity to cut up the protein before it is secreted away through the cytoplasmic membrane.

Coexpression of t-RNAs Specific for Rare Codons in hGH cDNA

The arginine codon AGG is extremely rare in S. carnosus genes, as it is used in only 0.8% of the arginine codons. The corresponding tRNA genes in S. carnosus have not yet been identified. Therefore, we expressed E. coli tRNAs for the AGG codon in S. carnosus . The E. coli genes argU and argW, which code the tRNAs for the codons AGG/AGA and AGG, had been cloned with their natural promoters and terminators into the plasmids pUBS520 or pSB101 (2, 16). The fragments were isolated as SalI-SpHI (argu) and XmaI-EcoRI (argw) fragments and cloned into the corresponding restriction cutting sites of the shuttle (transfer) vector pRB572 (4). The resulting plasmids pRBargU and pRBargW were transformed in S. carnosus (pTX-LipP-hGH2), which express hGH cDNA.

The protein patterns in the supernatant of the S. carnosus strains obtained are comparable with S. carnosus (pTX-LipP-hGH4) with increased production of the Lip-hGH protein and less truncated proteins. Therefore, the expression of rare tRNAs according to the invention has advantageous properties for the preparation of human proteins in S. carnosus , for example.

FIG. 1 . Expression of the Lip-hGH fusion in the plasmids pTX-LipP-hGH2 or pTX-LipP-hGH4. The gene fusion is made up of the DNA sequences coding for the signal peptide (SP) and the propeptide (PP) from Staphylococcus hyicus lipase and the hGH part of the mature protein (in black) and is transcribed by the xyl promoter (shown as a grey triangle). The hGH cDNA or the synthetic DNA sequence was inserted between the NsiI and the PstI cutting site. The 5′ end pf the hGH DNA sequence was modified to code the enterokinase cutting site ′DDDK′, followed by the mature hGH sequence, as shown underneath the gene.

FIG. 2 . Detection of the Lip-hGH fusion proteins in the S. carnosus supernatant. Equal amounts of the supernatant were separated by SDS-PAGE on Tris-glycine gels containing 15% acrylamide and then stained with Coomassie Blue. The traces 1-5 contain supernatants of S. carnosus strains containing the empty control vector pTX16 (15), a derivative of pTX15 (trace 1), the plasmids pTX-LipP-hGH4 with the improved hGH gene (trace 2), pTX-LipP-hGH2 with the hGH cDNA (trace 3), pTX-LipP-hGH2 plus pRBargU (trace 4), and pTX-LipP-hGH2 plus pRBargW (trace 5). Standard proteins and their molecular weights (kDa) are shown on the left-hand side. The supernatants were concentrated by precipitation with trichloroacetic acid. SDS page was carried out by standard methods from the prior art.

FIGS. 3A and 3B. Nucleotide and amino acid sequence of the hGH according to the invention (LipPhGH4, SEQ: ID-No.3).

Relevant restriction cutting sites are underlined once, the Shine-Dalgarno sequence is underlined twice. The signal peptidase 1 and the enterokinase cleavage site in the are underlined with a broken line. Only the regions between the BglII and NdeI site and between the NsiI and the PstI site had been synthetically produced and optimised. The remainder consists of the original sequence of the signal and propeptide of the lipase from Staphylococcus hyicus .

Literature

1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1990. Current protocols in molecular biology. John Wiley and Sons, Inc., New York, N.Y.

2. Brinkmann, U., R. E. Mattes, and P. Buckel. 1989. High-level expression of recombinant genes in Eschericia coli is dependent on the availability of the dnaY gene product. Gene. 85:109-114.

3. Brosius, J. 1989. Superpolylinkers in cloning and expression vectors. DNA. 8:759-777.

4. Brückner, R. 1992. A series of shuttle vectors for Bacillus subtilis and Escherichia coli . Gene. 122:187-192.

5. Cano, F., S. Liljeqvist, T. N. Nguyen, P. Samuelson, J. Y. Bonnefoy, S. Stahl, and A. Robert. 1999. A surface-displayed cholera toxin B peptide improves antibody response using food-grade staphylococci for mucosal subunit vaccine delivery. FEMS Immunol. Med. Microbiol. 25:289-298.

6. Del Tito, B. J., J. M. Ward, C. J. L. Hodgson, C. Gershater, H. Edwards, L. A. Wysocki, F. A. Watson, G. Sathe, and J. F. Kane. 1995. Effects of a minor isoleucyl tRNA on heterologous protein translation in Escherichia coli . J. Bacteriol. 177:7086-7091.

7. Demleitner, G., and F. Götz. 1994. Evidence for importance of the Staphylococcus hyicus lipase pro-peptide in lipase secretion, stability and activity. FEMS Microbiol. Lett. 121:189-198.

8. Gilbert, M., R. Morosoli, F. Shareck, and D. Kluepfel. 1995. Production and secretion of proteins by streptomycetes. Crit. Rev. Biotechnol. 15:13-39.

9. Götz, F. 1990. Development of a cloning system in Staphylococcus carnosus : Different processing of the Staphylococcus hyicus lipase in Staphylococcus camosus and Staphylococcus hyicus , p. 273-281. In R. P. Novick (ed.), Molecular biology of the staphylococci. VCH Publisher Inc., New York, N.Y.

10. Götz, F. 1990. Staphylococcus carnosus : a new host organism for gene cloning and protein production. J. Appl. Bacteriol. Symp. Suppl.:49S-53S.

11. Götz, F., and B. Schumacher. 1987. Improvements of protoplast transformation in Staphylococcus carnosus . FEMS Microbiol. Lett. 40:285-288.

12. Liljeqvist, S., F. Cano, T. N. Nguyen, M. Uhlen, A. Robert, and S. Stahl. 1999. Surface display of functional fibronectin-binding domains on Staphylococcus carnosus . FEBS Lett. 446:299-304.

13. Makrides, S. C. 1996. Strategies for achieving high-level expression of genes in Escherichia coli . Microbiol. Reviews. 60:512-538.

14. Nikoleit, K., R. Rosenstein, H. M. Verheij, and F. Götz. 1995. Comparative biochemical and molecular analysis of the Staphylococcus hyicus, Staphylococcus aureus and a hybrid lipase. Eur. J. Biochem 228:732-738.

15. Peschel, A., B. Ottenwälder, and F. Götz. 1996. Inducible production and cellular location of the epidermin biosynthetic enzyme EpiB using an improved staphylococcal expression system. FEMS Microbiol. Lett. 137:279-284.

16. Schenk, P. M., S. Baumann, R. Mattes, and H. H. Steinbiss. 1995. Improved high level expression system for eucaryotic genes in Escherichia coli using T7 RNA polymerase and rare Arg-tRNAs.

17. Thumm, G., and F. Götz. 1997. Studies on prolysostaphin processing and characterization of the lysostaphin immununity factor (Lif) of Staphylococcus simulans biovar staphylolyticus. Mol. Microbiol. 23:1251-1265.

18. Vance, M. L., and N. Mauras. 1999. Growth hormone therapy in adults and children. New Engl. J. Med. 341:1206-1216.

19. Wong, S. L. 1995. Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins. Curr. Opin. Biotechnol. 6:517-522.

<160> NUMBER OF SEQ ID NOS: 3

<210> SEQ ID NO 1

<211> LENGTH: 1383

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(2)

<223> OTHER INFORMATION: May be any nucleotide

<400> SEQUENCE: 1

nnagatctaa aggaggtaat tcatatgaaa gaaacaaaac atcaacacac at

#tttctatc     60

cgtaagtcgg cttatggtgc cgcgtcggtt atggtcgcat catgtatatt tg

#tcatcggt    120

gggggcgtgg cagaggcaaa tgattcgaca acacaaacaa cgacaccact ag

#aagtcgct    180

caaacgtcgc agcaagaaac acatacacat caaacacctg ttacatcatt ac

#atactgca    240

acacctgaac atgttgatga ctctaaagaa gcaacacctt tacctgaaaa ag

#cagagtca    300

ccaaaaaccg aagtgacagt tcaaccttca tcgcatacac aggaagtacc tg

#cgttacat    360

aaaaaaacac agcaacaacc ggcgtataag gataaaacgg taccagagtc aa

#cgatagca    420

tcaaagtcgg ttgaatcaaa taaagcaaca gaaaatgaga tgtcacctgt tg

#aacatcat    480

gcttcaaatg tggaaaaacg tgaagataga ttggagacta atgagacaac ac

#cgccatca    540

gtggaccgtg aatttagcca taaaatcatc aataatacgc acgtaaatcc aa

#aaacggat    600

ggacaaacaa acgttaatgt tgatacgaaa acgatagaca ccgtttcacc ga

#aagatgac    660

agaatagata cggcgcaacc gaaacaagtc gacgttccta aagaaaatac aa

#cggcacaa    720

aataaattta catcacaagc gagcgacaaa aaaccaacag taaaagatgc at

#caggtggt    780

gatgatgatg ataaatttcc aacaattccc ttaagtcgat tgttcgataa cg

#ctatgtta    840

cgtgcacata gattacacca gctagcattc gatacttatc aagaatttga gg

#aagcttac    900

atcccaaaag aacaaaagta tagcttttta caaaatccgc aaacatcatt at

#gtttctct    960

gaatcaattc caacacctag taaccgtgag gaaactcagc aaaaatcaaa tt

#tagaactt   1020

ttacgtatta gcttgttact tatacaatct tggttagaac cagttcaatt tt

#tacgttca   1080

gtattcgcaa atagtttagt ctatggtgct tcagactcta acgtatacga tt

#tattgaaa   1140

gacttagaag aaggaattca aacattaatg ggtcgtttgg aagatggttc ac

#caagaact   1200

ggccaaattt ttaaacaaac atatagcaaa ttcgatacta attcacataa cg

#atgacgca   1260

ttacttaaaa attacggttt attgtattgt tttcgtaaag atatggataa ag

#ttgaaaca   1320

ttcttacgca tagtacaatg ccgttctgtt gaaggatcat gtggttttta at

#gataactg   1380

cag

#

#

#           1383

<210> SEQ ID NO 2

<211> LENGTH: 448

<212> TYPE: PRT

<213> ORGANISM: Homo Sapiens

<400> SEQUENCE: 2

Met Lys Glu Thr Lys His Gln His Thr Phe Se

#r Ile Arg Lys Ser Ala

1               5

#                10

#                15

Tyr Gly Ala Ala Ser Val Met Val Ala Ser Cy

#s Ile Phe Val Ile Gly

20

#            25

#            30

Gly Gly Val Ala Glu Ala Asn Asp Ser Thr Th

#r Gln Thr Thr Thr Pro

35

#        40

#        45

Leu Glu Val Ala Gln Thr Ser Gln Gln Glu Th

#r His Thr His Gln Thr

50

#    55

#    60

Pro Val Thr Ser Leu His Thr Ala Thr Pro Gl

#u His Val Asp Asp Ser

65

#70

#75

#80

Lys Glu Ala Thr Pro Leu Pro Glu Lys Ala Gl

#u Ser Pro Lys Thr Glu

85

#                90

#                95

Val Thr Val Gln Pro Ser Ser His Thr Gln Gl

#u Val Pro Ala Leu His

100

#           105

#           110

Lys Lys Thr Gln Gln Gln Pro Ala Tyr Lys As

#p Lys Thr Val Pro Glu

115

#       120

#       125

Ser Thr Ile Ala Ser Lys Ser Val Glu Ser As

#n Lys Ala Thr Glu Asn

130

#   135

#   140

Glu Met Ser Pro Val Glu His His Ala Ser As

#n Val Glu Lys Arg Glu

145                 1

#50                 1

#55                 1

#60

Asp Arg Leu Glu Thr Asn Glu Thr Thr Pro Pr

#o Ser Val Asp Arg Glu

165

#               170

#               175

Phe Ser His Lys Ile Ile Asn Asn Thr His Va

#l Asn Pro Lys Thr Asp

180

#           185

#           190

Gly Gln Thr Asn Val Asn Val Asp Thr Lys Th

#r Ile Asp Thr Val Ser

195

#       200

#       205

Pro Lys Asp Asp Arg Ile Asp Thr Ala Gln Pr

#o Lys Gln Val Asp Val

210

#   215

#   220

Pro Lys Glu Asn Thr Thr Ala Gln Asn Lys Ph

#e Thr Ser Gln Ala Ser

225                 2

#30                 2

#35                 2

#40

Asp Lys Lys Pro Thr Val Lys Asp Ala Ser Gl

#y Gly Asp Asp Asp Asp

245

#               250

#               255

Lys Phe Pro Thr Ile Pro Leu Ser Arg Leu Ph

#e Asp Asn Ala Met Leu

260

#           265

#           270

Arg Ala His Arg Leu His Gln Leu Ala Phe As

#p Thr Tyr Gln Glu Phe

275

#       280

#       285

Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Ty

#r Ser Phe Leu Gln Asn

290

#   295

#   300

Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Il

#e Pro Thr Pro Ser Asn

305                 3

#10                 3

#15                 3

#20

Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Gl

#u Leu Leu Arg Ile Ser

325

#               330

#               335

Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Va

#l Gln Phe Leu Arg Ser

340

#           345

#           350

Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Se

#r Asp Ser Asn Val Tyr

355

#       360

#       365

Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gl

#n Thr Leu Met Gly Arg

370

#   375

#   380

Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Il

#e Phe Lys Gln Thr Tyr

385                 3

#90                 3

#95                 4

#00

Ser Lys Phe Asp Thr Asn Ser His Asn Asp As

#p Ala Leu Leu Lys Asn

405

#               410

#               415

Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Me

#t Asp Lys Val Glu Thr

420

#           425

#           430

Phe Leu Arg Ile Val Gln Cys Arg Ser Val Gl

#u Gly Ser Cys Gly Phe

435

#       440

#       445

<210> SEQ ID NO 3

<211> LENGTH: 1383

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Homo sapiens DNA sequence

# flanked by vectorial

DNA sequence

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(2)

<223> OTHER INFORMATION: May be any nucleotide

<400> SEQUENCE: 3

nnagatctaa aggaggtaat tcatatgaaa gaaacaaaac atcaacacac at

#tttctatc     60

cgtaagtcgg cttatggtgc cgcgtcggtt atggtcgcat catgtatatt tg

#tcatcggt    120

gggggcgtgg cagaggcaaa tgattcgaca acacaaacaa cgacaccact ag

#aagtcgct    180

caaacgtcgc agcaagaaac acatacacat caaacacctg ttacatcatt ac

#atactgca    240

acacctgaac atgttgatga ctctaaagaa gcaacacctt tacctgaaaa ag

#cagagtca    300

ccaaaaaccg aagtgacagt tcaaccttca tcgcatacac aggaagtacc tg

#cgttacat    360

aaaaaaacac agcaacaacc ggcgtataag gataaaacgg taccagagtc aa

#cgatagca    420

tcaaagtcgg ttgaatcaaa taaagcaaca gaaaatgaga tgtcacctgt tg

#aacatcat    480

gcttcaaatg tggaaaaacg tgaagataga ttggagacta atgagacaac ac

#cgccatca    540

gtggaccgtg aatttagcca taaaatcatc aataatacgc acgtaaatcc aa

#aaacggat    600

ggacaaacaa acgttaatgt tgatacgaaa acgatagaca ccgtttcacc ga

#aagatgac    660

agaatagata cggcgcaacc gaaacaagtc gacgttccta aagaaaatac aa

#cggcacaa    720

aataaattta catcacaagc gagcgacaaa aaaccaacag taaaagatgc at

#caggtggt    780

gatgatgatg ataaatttcc aacaattccc ttaagtcgat tgttcgataa cg

#ctatgtta    840

cgtgcacata gattacacca gctagcattc gatacttatc aagaatttga gg

#aagcttac    900

atcccaaaag aacaaaagta tagcttttta caaaatccgc aaacatcatt at

#gtttctct    960

gaatcaattc caacacctag taaccgtgag gaaactcagc aaaaatcaaa tt

#tagaactt   1020

ttacgtatta gcttgttact tatacaatct tggttagaac cagttcaatt tt

#tacgttca   1080

gtattcgcaa atagtttagt ctatggtgct tcagactcta acgtatacga tt

#tattgaaa   1140

gacttagaag aaggaattca aacattaatg ggtcgtttgg aagatggttc ac

#caagaact   1200

ggccaaattt ttaaacaaac atatagcaaa ttcgatacta attcacataa cg

#atgacgca   1260

ttacttaaaa attacggttt attgtattgt tttcgtaaag atatggataa ag

#ttgaaaca   1320

ttcttacgca tagtacaatg ccgttctgtt gaaggatcat gtggttttta at

#gataactg   1380

cag

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