Patent Application: US-42166195-A

Abstract:
an autoantigen identified as hp - 8 which is related to systemic lupus erythematosus . the hp - 8 antigen is expressed by a gene which was identified by immunoscreening of human placental cdna gt11 expression library with the monoclonal antibody 3e10 . the 3e10 antibody is a low - affinity anti - double - stranded dna autoantibody derived from the mrl murine models for human systemic lupus erythematosus .

Description:
the hp - 8 antigen in accordance with the present invention is defined as a protein or polypeptide which includes an epitope which is substantially homologous with the amino acid sequence set forth in seq id no . 4 . the protein or polypeptide will have a molecular weight of less than 100 kd . preferred proteins will have molecular weights on the order of 60 to 100 kd . to be considered substantially homologous , the amino acid sequence of the epitope of the protein or polypeptide must be at least about 90 % or more homologous with the amino acid sequence set forth in seq id no . 4 . proteins and polypeptides which fall under the definition of hp - 8 must bind 3e10 antibody . such hp - 8 proteins and polypeptides are also expected to bind calcium , hydroxyapatite and collagen . preferably , the hp - 8 antigen has the amino acid sequence set forth in seq id no . 6 or its amino acid sequence variants . most preferably , the hp - 8 antigen has the amino acid sequence set forth in seq id no . 6 . amino acid sequence variants of the hp - 8 antigen having the amino acid sequence set forth in seq id no . 6 fall into one or more of three classes : substitutional , insertional and deletional variants . these variants ordinarily are prepared by site specific mutagenesis of nucleotides in the dna encoding the variant , and thereafter expressing the dna in recombinant cell culture . however , variants having up to about 100 - 150 residues may be conveniently prepared using in vitro synthesis . amino acid substitutions are typically of single residues ; insertions will be on the order of about from 1 to 10 amino acids ; and deletions will range from about 1 to 30 amino acids . deletions and insertions preferably are made in adjacent pairs , i . e . a deletion of 2 residues or insertion of 2 residues . substitutions , deletions , insertions or any combination thereof may be combined to arrive at a final construct . obviously , the mutations made in the dna encoding the variant hp - 8 antigen must not place the sequence out of reading frame . insertional amino acid sequence variants of the hp - 8 antigen are those in which one or more amino acid residues are introduced into a predetermined site . most commonly , insertional variants are fusions of heterologous proteins or polypeptides to the amino or carboxyl terminus of the hp - 8 antigen . amino acid sequence variants of the hp - 8 antigen having the amino acid sequence set forth in seq id no . 6 must , as stated above , have an epitope which is substantially homologous ( i . e . 90 % homologous ) with the amino acid sequence set forth in seq id no . 4 . further , the entire protein or polypeptide will have a molecular weight of less than 100 kd , preferably between about 60 and 100 kd . in addition , the amino acid sequence variants of hp - 8 having the amino acid sequence set forth in seq id no . 6 must potentially bind 3e10 antibody , i . e . must be capable of binding the antibody to substantially the same extent as hp - 8 antigen having an epitope having the sequence set forth in seq id no . 4 . hp - 8 antigens in accordance with the present invention may be produced in accordance with any of the known processes for preparing polypeptides and proteins . it is preferred that the antigen be expressed in prokaryotic , eukaryotic or insect viral cells by recombinant means . an exemplary procedure for producing hp - 8 antigen is as follows : a commercially available cdna library was plated and screened according to manufacturers instructions . the cdna library was a human placental cdna gt11 expression library , catalog no : hl - 1075b ( clontech laboratories , palo alto , calif .). large 150 mm lb agar plates were used to plate and screen the library with mab 3e10 . 0 . 6 ml of plating bacteria ( y1090 ) was incubated with a proper dilution of lambda gt11 phage and absorbed to the cells at 370 ° c . for 15 minutes . 7 . 5 ml of lb soft agarose was added to the culture and quickly poured onto the plates and incubated at 42 ° c . for 3 . 5 hours . plates were removed and overlaid with a dry nitrocellulose filter previously saturated in 10 mm isopropyl - 1 - thio - b - d - galactoside ( iptg ). the plates were incubated for an additional 3 . 5 hours at 37 ° c . filters were removed and rinsed in 50 mm tris ( ph 7 . 9 ), 150 mm nacl , 0 . 05 % tween ( tbst ) buffers . filters were incubated with 10 ug / ml of mab in tbst buffer for 3 hours at room temperature . following incubation filters were washed in three changes of buffer a for 3 minutes each . detection of bound antibody was done using the clik ii immunoscreening kit ( clontech laboratories , palo alto , calif ., catalog number : k1004 - 2 ). detection of bound antibody used an alkaline phosphatase conjugate . filters were incubated with goat - anti - mouse conjugate ( 2 ul ) in 5 ml of buffer a for 30 minutes . following incubation the filters were washed 3 times with 50 ml of buffer a ( 10 minutes each wash ). an additional wash was done in buffer c for 10 minutes . detection was performed by addition of 25 ul nbt ( 100 mg / ml ) and 12 ul 5 - bromo - 4 - chloro - 3 - indolyl phosphate ( bcip ) ( 100 mg / ml ). filters were incubated until signals became visible under reduced illumination . the reaction was terminated by washing in 1 mm edta and positives selected . six positives were identified in the screen and one was determined to be a true positive following secondary and tertiary rescreening using dilution cloning . the positive , designated hp - 8 , was screened against normal human sera as a negative control indicating the validity of the 3e10 reactivity . details of the preparation of the buffers are described in the clontech handbook ( 1992 ). the lambda phage was grown up on plates according to protocols supplied from clontech ( pgs . 20 - 22 , clontech protocol handbook 1992 ). isolated dna was obtained and eco r1 digested using standard methods described in maniatis , t . et al ., ( 1989 ) molecular cloning : a laboratory manual . 2nd ed . ( cold spring harbor laboratory press ; plainview , n . y .). an insert of approximately 200 bases was resolved when electrophoresed in a 1 % agarose gel in tbe . the insert was pcr amplified according to manufacturers instructions and subcloned into pcr ii . ( invitrogen , san diego , calif ., catalog k2000 - 01 ). the subcloned fragment was retained as a hard copy template for subsequent expression cloning . additional pcr amplification of insert was performed to generate material for subcloning into pbluescript for dna sequencing . ( stratagene , la jolla , calif ., catalog number : 212205 ). the double - stranded pbluescript pskii + plasmids containing the hp - 8 specific clone fragment were grown and dna harvested using the qiagen column purification system ( qiagen corp ., chatsworth , calif ., catalog number : 12162 ). t3 and t7 primers were used to sequence the cdna . procedures were followed using standard cycle sequencing conditions recommended by the manufacturers ( abi , foster city , calif ., catalog number : 401384 ). the nucleotide sequence and corresponding amino acid sequence are set forth in seq id no : 1 and seq id no : 2 , respectively . the nucleotide sequence set forth in seq id no . 1 has a length of 154 bp . the corresponding amino acid sequence ( seq id nos . 1 & amp ; 2 ) is 51 amino acids in length . however , as is well known , cdna clones from libraries often contain artifactual dna sequence inserts at the termini of the sequence actually corresponding to mrna . in accordance with this , it was determined that the actual size of the hp - 8 insert was 132 bp and the corresponding amino acid sequence was 44 amino acids ( seq id nos . 3 & amp ; 4 ). in order to obtain full length coding sequences for hp8 , a human fetal brain cdna expression library in lambda zap ii ( stratagene , cat . # 936206 ) was screened by dna hybridization to the 132 bp hp8 cdna insert isolated from the immunoscreen . brain was chosen as tissue source because northern hybridization results indicated that hp - 8 was highly expressed in this tissue . hybridizing clones were purified and rescued to pbluescript skii + ( stratagene ) plasmids containing hp8 hybridizing cdna inserts . clones were first sequenced from the ends using t3 and t7 promoter primers and with hp - 8 specific primers to verify authenticity as an overlapping hp - 8 cdna sequence . the sequence of the largest overlapping clone , hfb4 - 1 ( 1790 bp ) was determined in its entirety along both strands by using nested primers , assembled using gel software ( intelligenetics ), and is included in fig1 . translation of the nucleotide sequence in all reading frames revealed a single orf encoding a 459 amino acid sequence . hfb4 - 1 appears to be a truncated cdna clone ( deficient in 5 &# 39 ; sequences ), that encodes the c - terminal 459 amino acids of hp - 8 , and approximately 400 bp of 3 &# 39 ; untranslated ( ut ) nucleotide sequence . a putative poly - a addition site is just upstream from the 3 &# 39 ; end of the clone , indicating that hfb4 - 1 contains the authentic 3 &# 39 ; end of the mrna . southern blot analyses , using genomic dna ( human , monkey , rat , mouse , dog , cow , rabbit , chicken , and yeast ) ( clontech , and bios laboratories ) revealed a simple pattern of hfb4 - 1 hybridization , indicative of single copy or unique sequences . this sequence appears to be highly conserved , as hybridizing sequences were detected in all species tested . chromosomal mapping of hfb4 - 1 sequences was determined by somatic cell hybrid southern blot analyses . hybridization of hfb4 - 1 sequences to the dna from a panel of human - hamster cell lines ( bios laboratories ) showed mapping to human chromosome 4 . human osteonectin has been mapped to human chromosome 5 , indicating that hp - 8 is not encoded by the same gene as osteonectin , and also that the results of these southern analyses were not due to cross hybridization with human osteonectin sequences . to circumvent the limitations of using a cdna library to isolate 5 &# 39 ; sequences from large mrnas , a 5 &# 39 ; race ( rapid amplification of cdna ends ) procedure was employed using a commercially available kit ( clontech , palo alto , calif .). using a 5 &# 39 ; directed hfb4 - 1 ( largest hp - 8 containing cdna clone )- specific antisense primer ( hfb4 - 1 - race - 1a ) and human brain poly - a rna ( clontech , palo alto , calif .) as template , an hp - 8 specific 5 &# 39 ; cdna was synthesized to which a 5 &# 39 ; &# 34 ; tag &# 34 ; anchor primer sequence was ligated . using a nested hp - 8 antisense primer ( hfb4 - 1 - race - 2a ), and a &# 34 ; tag &# 34 ; sequence sense primer , the hp - 8 5 &# 39 ; sequences were amplified by polymerase chain reaction ( pcr ). subsequent &# 34 ; shotgun &# 34 ; litigation of the amplification product into the pcr2000 vector ( ta cloning system , invitrogen , san diego , calif .) and colony hybridization to another nested primer ( hfb4 - 1 - race - 3a ) allowed isolation of 5 &# 39 ; hp - 8 cdna containing clones ( race clones ). dna was purified from these candidates , and the inserts were characterized by restriction endonuclease ( bamhi or ecori ) digestion , to release the insert , followed by size fractionation by agarose gel electrophoresis , transfer to nitrocellulose and subsequent southern blot hybridization to hp - 8 primer 3a ( sambrook , j ., fritsch , e . f ., and maniatis , t . ( 1989 ) molecular cloning : a laboratory manual , second edition , cold spring harbor laboratory press , cold spring harbor , n . y .). clones containing the largest &# 34 ; hfb4 - 1 - race - 3a &# 34 ; hybridizing inserts were subjected to dna sequence analyses . cycle sequencing ( applied biosystems , foster city , calif .) of the inserts using polylinker primers and subsequent internal sequence specific primers ( primer walking ) was performed using an applied biosystems automated dna sequencer . sequences were edited using the applied biosystems seqed program and assembled using gel software ( intelligenetics , mountain view , calif .). race clone # 9 ( ta - hb - race9 ) contained hp - 8 cdna sequence that overlapped with hfb4 - 1 sequences and encoded a continguous open reading frame ( orf ). this clone is represented schematically in fig1 and the sequence is included in fig2 a - 1 through 2a - 8 . because the sequence of race clone # 9 did not contain the atg start codon , the 5 &# 39 ; race procedure was repeated , using primers designed to upstream sequences in race clone # 9 ( primers hfb4 - 1 - race 4a and hfb4 - 1 - race - 5a ), and screened with a race clone # 9 upstream primer ( hfb4 - 1 - race - 6s ). one isolate , race clone # 6 ( ta - hb6 ) contained race clone # 9 overlapping sequence , and encoded the putative atg start codon , preceded by an in - frame stop codon . race clone # 6 is depicted schematically in fig1 and the sequence is included in fig2 a - 1 through 2a - 8 . the first 17 amino acids of the deduced sequence appear to encode a signal sequence characteristic of secreted proteins ( von heijine , g . ( 1983 ) eur . j . biochem . 133 : 17 ). the predicted amino acid sequence shows some colinearity with the rat sc1 protein , but the percent identity is not as strong as in the &# 34 ; osteonectin domain &# 34 ;, indicating that sc1 and hp - 8 are two distinct and different proteins . a multiple tissue northern blot was obtained from clontech laboratories , palo alto , calif . ( catalog number : 7760 - 1 ) and hybridized to 32p labelled cdna insert from hp - 8 . the probe was prepared according to manufacturers instructions ( brl , gaithersburg , md ., catalog number : 8187 - sa ) at high specific activity . hybridization conditions were performed as described in the clontech handbook for product number 7760 - 1 . washed filters were air dried and exposed to xr - 5 kodak x - ray film for 18 hours at - 70 ° c . the epitope of the hp - 8 antigen ( i . e . seq id no : 4 ) is about 60 - 80 percent homologous with various proteins and polypeptides which belong to the osteonectin family ( see p . t . russell et al ., the osteonectin famil of proteins , j . biochem ., vol . 20 , no . 7 , pp . 653 - 660 , 1988 ). specific examples of related osteonectin proteins are osteonectin / bm401 sparc and sc1 . these specific osteonectins are described in the following three references : 1 . j . h . mcvey et al ., characterization of the mouse sparc / osteonectin gene , jour . biological chem ., vol . 263 , issue of august 15 , pp . 11111 - 11116 , 1988 ; 2 . j . engel et al ., calcium binding domains and calcium - induced conformational transition of sparc / bm - 40 / osteonectin , an extracellular glycoprotein expressed in mineralized and nonmineralized tissue , biochemistry , 1987 , vol . 26 , 6958 - 6965 ; and 3 . i . g . johnston , et al ., molecular cloning of sc1 : a putative brain extracellular matrix glycoprotein showing partial similarity to osteonectin / bm40 / sparc , neuron , vol . 2 , 165 - 176 , jan . 1990 . the deduced amino acid sequence of hfb4 - 1 was used to search the pir 38 and swiss - prot 26 databases using the fastdb program ( intelligenetics ). the deduced amino acid sequence of hfb4 - 1 showed an overall sequence identity of 62 % with rat sc1 , and 49 % and 50 % identity with human and murine osteonectin , respectively . the final 200 residues of the sc1 protein ( 634 aa ), as derived from cdna sequence shows approximately 65 % sequence identity with the final 200 residues of osteonectin ( 304 aa ). hfb4 - 1 amino acid sequence is also most similar to sc1 and to osteonectin / sparc / bm40 throughout the c - terminal 200 residues . dna sequences which code on expression for hp - 8 antigen may be inserted into appropriate expression vectors for expression in prokaryotic , eukaryotic or insect viral cells . a wide variety of expression vectors are available and may be used in conventional procedures to transform competent host cells for expression and isolation of the hp - 8 antigen . methods for preparing gene sequences , inserting the sequences into expression vectors , transforming competent hosts and growing the host in culture for production of products are disclosed in u . s . pat . nos . 4 , 710 , 473 ; 4 , 711 , 843 ; and 4 , 713 , 339 . the hp - 8 antigen can be used to generate antibodies . the hp - 8 antigen can be used in any of the conventional procedures involving administering an antigen to a host animal in order to raise antibodies . the administration protocols , including dosage levels , administration schedules and isolation and recovery of antibodies from the host animal are all well known in the art . the hp - 8 antigen is used in the same manner as any other antigen to elicit the production of antibodies in a host animal . the hp - 8 antigen includes epitopes which bind 3e10 antibodies and therefore will be useful in investigating the etiology of sle . in addition , hp - 8 will be useful in developing therapeutic rational drug designs which will be effective in treating sle and other related connective tissue diseases such as rheumatoid arthritis . all of the united states patents , literature references and methodology handbooks set forth in this specification are hereby incorporated by reference . having thus described exemplary embodiments of the present invention , it will be understood by those skilled in the art that the above disclosures are exemplary only and that the present invention is not limited to the embodiments as disclosed herein , but is only limited by the following claims . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 6 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 154 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vi ) original source :( a ) organism : homo sapiens ( ix ) feature :( a ) name / key : cds ( b ) location : 2 .. 154 ( xi ) sequence description : seq id no : 1 : ggaattcgggtttccctgtgtctgcgaggatccagtgacttgtcct46glupheglypheprocysvalcysgluaspprovalthrcyspro151015ccaacaaaaccccttgatcaagtttgtggcactgacaatcagacctat94prothrlysproleuaspglnvalcysglythraspasnglnthrtyr202530gctagttcctgtcatctattcgctactaaatgcagactggaggggacc142alasersercyshisleuphealathrlyscysargleugluglythr354045aaaaaggccccc154lyslysalapro50 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 51 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 2 : glupheglypheprocysvalcysgluaspprovalthrcyspropro151015thrlysproleuaspglnvalcysglythraspasnglnthrtyrala202530sersercyshisleuphealathrlyscysargleugluglythrlys354045lysalapro50 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 132 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( ix ) feature :( a ) name / key : cds ( b ) location : 1 .. 132 ( xi ) sequence description : seq id no : 3 : tgtgtctgccaggatccagtgacttgtcctccaacaaaaccccttgat48cysvalcysglnaspprovalthrcysproprothrlysproleuasp556065caagtttgtggcactgacaatcagacctatgctagttcctgtcatcta96glnvalcysglythraspasnglnthrtyralasersercyshisleu707580ttcgctactaaatgcagactggaggggaccaaaaag132phealathrlyscysargleugluglythrlyslys859095 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 44 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 4 : cysvalcysglnaspprovalthrcysproprothrlysproleuasp151015glnvalcysglythraspasnglnthrtyralasersercyshisleu202530phealathrlyscysargleugluglythrlyslys3540 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 2465 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : cdna ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : n - terminal ( vi ) original source :( a ) organism : homo sapiens ( ix ) feature :( a ) name / key : cds ( b ) location : 61 .. 2054 ( xi ) sequence description : seq id no : 5 : caacttcaatttttctgcagtggctctgggtccagccccttacttaaagatctggaaagc60atgaagactgggctttttttcctatgtctcttgggaactgcagctgca108metlysthrglyleuphepheleucysleuleuglythralaalaala45505560atcccgacaaatgcaagattattatctgatcattccaaaccaactgct156ileprothrasnalaargleuleuserasphisserlysprothrala657075gaaacggtagcacccgacaacactgcaatccccagtttaagggctgaa204gluthrvalalaproaspasnthralaileproserleuargalaglu808590gatgaagaaaatgaaaaagaaacagcagtatccacagaagacgattcc252aspglugluasnglulysgluthralavalserthrgluaspaspser95100105caccataaggctgaaaaatcatcagtactaaagtcaaaagaggaaagc300hishislysalaglulysserservalleulysserlysglugluser110115120catgaacagtcagcagaacagggcaagagttctagccaagagctggga348hisgluglnseralagluglnglylysserserserglngluleugly125130135140ttgaaggatcaagaggacagtgatggtgacttaagtgtgaatttggag396leulysaspglngluaspseraspglyaspleuservalasnleuglu145150155tatgcaccaactgaaggtacattggacataaaagaagatatgagtgag444tyralaprothrgluglythrleuaspilelysgluaspmetserglu160165170cctcaggagaaaaaactctcagagaacactgattttttggctcctggt492proglnglulyslysleusergluasnthrasppheleualaprogly175180185gttagttccttcacagattctaaccaacaagaaagtatcacaaagaga540valserserphethraspserasnglnglngluserilethrlysarg190195200gaggaaaaccaagaacaacctagaaattattcacatcatcagttgaac588glugluasnglngluglnproargasntyrserhishisglnleuasn205210215220aggagcagtaaacatagccaaggcctaagggatcaaggaaaccaagag636argserserlyshisserglnglyleuargaspglnglyasnglnglu225230235caggatccaaatatttccaatggagaagaggaagaagaaaaagagcca684glnaspproasnileserasnglyglugluglugluglulysglupro240245250ggtgaagttggtacccacaatgataaccaagaaagaaagacagaattg732glygluvalglythrhisasnaspasnglngluarglysthrgluleu255260265cccagggagcatgctaacagcaagcaggaggaagacaatacccaatct780proarggluhisalaasnserlysglnglugluaspasnthrglnser270275280gatgatattttggaagagtctgatcaaccaactcaagtaagcaagatg828aspaspileleuglugluseraspglnprothrglnvalserlysmet285290295300caggaggatgaatttgatcagggtaaccaagaacaagaagataactcc876glngluaspglupheaspglnglyasnglngluglngluaspasnser305310315aatgcagaaatggaagaggaaaatgcatcgaacgtcaataagcacatt924asnalaglumetgluglugluasnalaserasnvalasnlyshisile320325330caagaaactgaatggcagagtcaagagggtaaaactggcctagaagct972glngluthrglutrpglnserglngluglylysthrglyleugluala335340345atcagcaaccacaaagagacagaagaaaagactgtttctgaggctctg1020ileserasnhislysgluthrgluglulysthrvalserglualaleu350355360ctcatggaacctactgatgatggtaataccacgcccagaaatcatgga1068leumetgluprothraspaspglyasnthrthrproargasnhisgly365370375380gttgatgatgatggcgatgatgatggcgatgatggcggcactgatggc1116valaspaspaspglyaspaspaspglyaspaspglyglythraspgly385390395cccaggcacagtgcaagtgatcactacttcatcccaagccaggccttt1164proarghisseralaserasphistyrpheileproserglnalaphe400405410ctggaggccgagagagctcaatccattgcctatcacctcaaaattgag1212leuglualagluargalaglnserilealatyrhisleulysileglu415420425gagcaaagagaaaaagtacatgaaaatgaaaatataggtaccactgag1260gluglnargglulysvalhisgluasngluasnileglythrthrglu430435440cctggagagcaccaagaggccaagaaagcagagaactcatcaaatgag1308proglygluhisglnglualalyslysalagluasnserserasnglu445450455460gaggaaacgtcaagtgaaggcaacatgagggtgcatgctgtggattct1356glugluthrsersergluglyasnmetargvalhisalavalaspser465470475tgcatgagcttccagtgtaaaagaggccacatctgtaaggcagaccaa1404cysmetserpheglncyslysargglyhisilecyslysalaaspgln480485490cagggaaaacctcactgtgtctgccaggatccagtgacttgtcctcca1452glnglylysprohiscysvalcysglnaspprovalthrcyspropro495500505acaaaaccccttgatcaagtttgtggcactgacaatcagacctatgct1500thrlysproleuaspglnvalcysglythraspasnglnthrtyrala510515520agttcctgtcatctattcgctactaaatgcagactggaggggaccaaa1548sersercyshisleuphealathrlyscysargleugluglythrlys525530535540aaggggcatcaactccagctggattattttggagcctgcaaatctatt1596lysglyhisglnleuglnleuasptyrpheglyalacyslysserile545550555cctacttgtacggactttgaagtgattcagtttcctctacggatgaga1644prothrcysthraspphegluvalileglnpheproleuargmetarg560565570gactggctcaagaatatcctcatgcagctttatgaagccaactctgaa1692asptrpleulysasnileleumetglnleutyrglualaasnserglu575580585cacgctggttatctaaatgagaagcagagaaataaagtcaagaaaatt1740hisalaglytyrleuasnglulysglnargasnlysvallyslysile590595600tacctggatgaaaagaggcttttggctggggaccatcccattgacctt1788tyrleuaspglulysargleuleualaglyasphisproileaspleu605610615620ctcttaagggactttaagaaaaactaccacatgtatgtgtatcctgtg1836leuleuargaspphelyslysasntyrhismettyrvaltyrproval625630635cactggcagtttagtgaacttgaccaacaccctatggatagagtcttg1884histrpglnphesergluleuaspglnhisprometaspargvalleu640645650acacattctgaacttgctcctctgcgagcatctctggtgcccatggaa1932thrhissergluleualaproleuargalaserleuvalprometglu655660665cactgcataacccgtttctttgaggagtgtgaccccaacaaggataag1980hiscysilethrargphephegluglucysaspproasnlysasplys670675680cacatcaccctgaaggagtggggccactgctttggaattaaagaagag2028hisilethrleulysglutrpglyhiscyspheglyilelysgluglu685690695700gacatagatgaaaatctcttgttttgaacgaagattttaaagaact2074aspileaspgluasnleuleuphe705caactttccagcatcctcctctgttctaaccacttcagaaatatatgcagctgtgatact2134tgtagatttatatttagcaaaatgttagcatgtatgacaagacaatgagagtaattgctt2194gacaacaacctatgcaccaggtatttaacattaactttggaaacaaaaatgtacaattaa2254gtaaagtcaacatatgcaaaatactgtacattgtgaacagaagtttaattcatagtaatt2314tcactctctgcattgacttatgagataattaatgattaaactattaatgataaaaataat2374gcatttgtattgttcataatatcatgtgcacttcaagaaaatggaatgctactcttttgt2434ggtttacgtgtattattttcaatatcttaat2465 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 664 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 6 : 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