Patent Application: US-70780200-A

Abstract:
the invention features a method for identifying a cdna nucleic acid encoding a mammalian protein having a signal sequence , which method includes the following steps : providing library of mammalian cdna ; ligating the library of mammalian cdna to dna encoding alkaline phosphatase lacking both a signal sequence and a membrane anchor sequence to form ligated dna ; transforming bacterial cells with the ligated dna to create a bacterial cell clone library ; isolating dna comprising the mammalian cdna from at least one clone in the bacterial cell clone library ; separately transfecting dna isolated from clones in step into mammalian cells which do not express alkaline phosphatase to create a mammalian cell clone library wherein each clone in the mammalian cell clone library corresponds to a clone in the bacterial cell clone library ; identifying a clone in the mammalian cell clone library which expresses alkaline phosphatase ; identifying the clone in the bacterial cell clone library corresponding to the clone in the mammalian cell clone library identified in step ; and isolating and sequencing a portion of the mammalian cdna present in the bacterial cell library clone identified in step to identify a mammalian cdna encoding a mammalian protein having a signal sequence .

Description:
1 . preparation of a randomly primed cdna library using cdna prepared from mrna extracted from mammalian cells or tissue . the cdna is inserted into a mammalian expression vector adjacent to a cdna encoding placental alkaline phosphatase which lacks a secretory signal . 5 . assay of supernatants from the transfected mammalian cells for alkaline phosphatase activity . 6 . isolation and sequencing of plasmid dna clones registering a positive score in the alkaline phosphatase assay . 7 . isolation of full length cdna clones of novel proteins having a signal sequence . the mammalian cdna used to create the cdna library can be prepared using any known method . generally , the cdna is produced from mrna . the mrna can be isolated from any desired tissue or cell type . for example , peripheral blood cells , primary cells , tumor cells , or other cells may be used as a source of mrna . the expression vector harboring the modified alkaline phosphatase gene can be any vector suitable for expression of proteins in mammalian cells . the mammalian cells used in the transfection step can be any suitable mammalian cells , e . g ., cho cells , mouse l cells , hela cells , vero cells , mouse 3t3 cells , and 293 cells . described below is a specific example of the method of the invention . also described below are two genes , one known and one novel , identified using this method . a cdna library was prepared using ptrap3 , a mammalian expression vector containing a cdna encoding human placental alkaline phosphatase ( ap ) lacking a signal sequence ( fig1 and fig2 seq id no : 1 ). when ptrap3 is transfected into a mammalian cell line , such as cos7 cells , ap protein is neither expressed nor secreted since the ap cdna of ptraap3 does not encode a translation initiating methionine , a signal peptide , or a membrane anchor sequence . fig3 ( seq id no : 2 ) provides the amino acid sequence of naturally occurring ap . fig4 ( seq id no : 3 ) provides the amino acid sequence of the form of ap encoded by ptrap3 . however , insertion of a cdna encoding a signal peptide sequence into ptrap3 such that the signal sequence within the cdna is fused to and in frame with ap , facilities both the expression and secretion of ap protein upon transfection of the dna into cos7 cells or other mammalian cells . the presence of ap activity in the supernatants of transfected cos7 cells therefore indicates the presence of a signal sequence in the cdna of interest . cdna for ligation to the ptrap3 vector was prepared from messenger rna isolated from human fetal brain tissue ( clontech , palo alto , calif . : catalog # 6525 - 1 ) by a modification of a commercially available “ zap cdna synthesis kit ” ( stratagene ; la jolla , calif . : catalog # 200401 ). synthesis of cdna involved the following steps . ( a ) single stranded cdna was synthesized from 5 μg of human fetal brain messenger rna using a random hexamer primer incorporating a xhol restriction site ( underlined ); 5 ′- ctgactcgagnnnnnn - 3 ′ ( seq id no : 4 ). this represented a deviation from the stratagene protocol and resulted in a population of randomly primed cdna molecules . random priming was employed rather than the oligo d ( t ) priming method suggested by stratagene in order to generate short cdna fragments , some of which would be expected to be mrnas that encode signal sequences . ( b ) the single stranded cdna generated in step ( a ) was rendered double stranded , and dna linkers containing a free ecor1 overhang were ligated to both ends of the double stranded cdnas using reagents and protocols from the stratagene zap cdna synthesis kit according to the manufacturer &# 39 ; s instructions . ( c ) the linker - adapted double - stranded cdna generated in step ( b ) was digested with xhoi to generate a free xhoi overhang at the 3 ′ end of the cdnas using reagents from the stratagene zap cdna synthesis kit according to the manufacturers instructions . ( d ) linker - adapted double - stranded cdnas were size selected by gel filtration through sephacryl ™ s - 500 cdna size fractionation columns ( gibco brl ; bethesda , md . : catalog # 18092 - 015 ) according to the manufacturers instructions . ( a ) size selected , double - stranded cdnas containing a free ecor1 overhang at the 5 ′ end and a free xhoi overhang at the 3 ′ end were ligated to the ptrap3 backbone which had been digested with ecor1 and xhol and purified from the small , released fragment by agarose gel electrophoresis . ( f ) ligated plasmid dnas were transformed into e . coli strain dh10b by electroporation . this process resulted in a library of cdna clones composed of several million random primed cdnas ( some of which will encode signal sequences ) prepared from human fetal brain messenger rna , fused to the ap reporter cdna , in the mammalian expression vector ptrap3 . next , the transformed bacterial cells were plated , and individual clones were identified . a sample of transformed e . coli containing the random primed human fetal brain cdna library described in step 1 was plated for growth as individual colonies , using standard procedures . each e . coli colony contained an individual cdna clone fused to the ap reporter in the ptrap3 expression vector . approximately 20 , 000 such e . coli colonies were plated , representing approximately 0 . 5 % of the total cdna library . next , e . coli colonies were picked from the plates and inoculated into deep well 96 well plates containing 1 ml of growth medium prepared by standard procedures . colonies were picked from the plates and e . coli cultures were grown overnight by standard procedures . each plate was identified by number . within each plate , each well contained an individual cdna clone in the ptrap vector identified by well position . finally , plasmid dna was extracted from the overnight e . coli cultures using a semi - automated 96 - well plasmid dna miniprep procedure , employing standard procedures for bacterial lysis , genomic dna precipitation and plasmid dna purification . ( a ) e . coli were centrifuged for 20 minutes using a beckman centrifuge at 3200 rpm . ( b ) supernatant was discarded and e . coli pellets were resuspended in 130 μl wp1 ( 50 mn tris ( ph 7 . 5 ), 10 mn edta , 100 μg / ml rnase a ) resuspension solution using a titerteck multidrop ™ apparatus . ( d ) 130 μl wp2 ( 0 . 2 m naoh , 0 . 5 % sds ) lysing solution was added to each well , and the samples were mixed by vortexing for 5 seconds . ( e ) 130 μl wp3 ( 125 mm potassium acetate , ph 4 . 8 ) neutralizing solution was added to each well , and the samples were mixed by vortexing for 5 seconds . ( f ) samples were placed on ice for 15 minutes , mixed by vortexing for 5 seconds , and recentrifuged for 10 minutes at 3200 rpm in a beckman centrifuge . ( g ) supernatant ( crude dna extract ) was transferred from each well of each 96 well plate into a 96 well filter plate ( polyfiltronics ) using a tomtec / quadra 96 ™ transfer apparatus . ( h ) 480 μl of wizard ™ midiprep dna purification resin ( promega ) was added to each well of each plate containing crude dna extract using a titertek multidrop apparatus and the samples were left for 5 minutes . ( i ) each 96 well filter plate was placed on a vacuum housing ( polyfiltronics ) and the liquid in each well was removed by suction generated by vacuum created with a lab port vacuum pump . ( j ) the wizard midiprep dna purification resin in each well ( to which plasmid dna was bound ) was washed four times with 600 μl of wizard wash ™. ( k ) plates were centrifuged for 5 minutes to remove excessive moisture from the wizard midiprep dna purification resin . ( l ) purified plasmid dnas were eluted from the wizard midiprep dna purification resin into collection plates by addition of 50 μl deionized water to each well using a multidrop 8 channel pipette , incubation at room temperature for 15 minutes , and centrifugation for 5 minutes ( 3200 rpm , beckman centrifuge ). this process resulted in preparation of plasmid dna contained in 96 well plates with each well containing an individual cdna clone ligated in the ptrap expression vector . individual clones were identified by plate number and well position . to determine which of the cdna clones contained within the cdna library encoded functional signal peptides , individual plasmid dna preparations were transfected into cos7 cells as follows . for each 96 well plate of dna preparations , one 96 well tissue culture plate containing approximately 10 , 000 cos7 cells per well was prepared using standard procedures . immediately prior to dna transfection , the cos7 cell culture medium in each well of each 96 well plate was replaced with 80 ul of optimem ( gibco - brl ; catalog # 31985 - 021 ) containing 1 μl of lipofectamine ( gibco - brl ) and 2 μl ( approximately 100 - 200 ng ) of dna prepared as described above . thus , each well of each 96 well plate containing cos7 cells received dna representing one individual cdna clone from the cdna library in ptrap3 . the cos7 cells were incubated with the opti - mem / lipofectamine / dna mixture overnight to allow transfection of cells with the plasmid dnas . after overnight incubation , the transfection medium was removed from the cells and replaced with 80 μl fresh medium composed of opti - mem + 1 % fetal calf serum . cells were incubated overnight . the secreted alkaline phosphatase activity of the transfected cos7 cells was measured as follows . samples ( 10 μl ) of supernatants from the transfected cos7 cells were transferred from each well of each 96 well plate into one well of a microfluor scintillation plate ( dynatech : location catalog # 011 - 010 - 7805 ). ap activity in the supernatants was determined using the phospha - light kit ( tropix inc . ; catalog # bp300 ). ap assays wore performed according to the manufacturer &# 39 ; s instruction using a wallace micro - beta scintillation counter . the individual plasmid dnas scoring positive in the cos7 cell ap secretion assay were analyzed further by dna sequencing using standard procedures . the resulting dna sequence information was used to perform blast sequence similarity searches of nucleotide protein databases to ascertain whether the clone in question encodes either 1 ) a known secreted or membrane - associated protein possessing a signal sequence , or 2 ) a putative novel , secreted or membrane - associated protein possessing a putative novel signal sequence . identification of the protein tyrosine phosphatase sigma ( ptpσ ) signal sequence by mammalian signal peptide trap employing the method described in example 1 , a cdna clone designated ethb005c07 was found to score positive in the cos7 cell transfection ap assay . blast similarity searching with the dna sequence from this clone identified ethb005c07 as a cdna encoding the signal sequence of protein tyrosine phosphatase sigma ( ptpσ ), a previously described protein that is well established in the scientific literature to be a transmembrane protein ( pulido et al ., proc . nat &# 39 ; l acad . sci . usa 92 : 11686 , 1995 ). identification of a novel immunoglobulin domain containing protein by mammalian signal peptide trap employing the method described in example 1 , a cdna clone designated ethb0018f2 was found to score positive in the cos7 cell transfection ap assay . dna sequencing revealed that ethb0018f2 harbors a 1455 base pair cdna having a single open reading frame commencing at nucleotide 55 and continuing to nucleotide 1455 . thus , the ethb0018f2 cdna encodes a 467 amino acid open reading frame ( fig5 seq id no : 5 ) fused to the ap reporter . inspection of the ethb0018f2 protein sequence revealed the presence of a putative signal sequence between amino acids 1 to 20 , predicted by the signal peptide prediction algorithm , signal p ( von heijne , nucleic acids . reg . 14 : 4683 - 90 , 1986 ). thus , ethb0018f2 encodes a partial clone of a novel putative secreted / membrane protein . blast similarity searching of nucleic acid and protein databases with the ethb0018f2 dna sequence from this clone revealed similarity to a family of proteins known to contain a protein motif referred to as an immunoglobulin of igg domain . further visual inspection of the ethb0018f2 protein sequence resulted in the identification of 5 consecutive igg repeats , defined by a conserved spacing of cysteine , tryptophan , tyrosine , and cysteine residues ( fig5 ). fig6 is a depiction of a protein sequence alignment between clone ethb0018f2 ( referred to as 8f2 ) and seven related proteins known to contain igg domains that are also known to be expressed in the brain . these proteins are rat neural adhesion molecule f3 ( d38492 ), drosophila neuroglian ( p20241 ), human neural adhesion molecule l1 ( p32004 ), chick neural adhesion molecule related ( p35331 ), human axonin 1 ( q02246 ), rat neural adhesion molecule big1 ( u11031 ) and chicken neurofascin ( x65224 ). given this sequence similarity , it is likely that clone ethb0018f2 represents a partial cdna cone representing a novel protein , expressed in the brain , which contains multiple , consecutive igg domains . specifically , since the closest relatiaves of clone ethb0018f2 are believed to function as neural adhesion molecules , it is likely that clone ethb0018f2 represents a partial cdna clone of a novel neural adhesion molecule . it is to be understood that while the invention has been described in conjunction with the detailed description thereof , that the foregoing description is intended to illustrate and not limit the scope of the invention , which is defined by the scope of the appended claims . ala ala lys asn leu ile ile phe leu gly asp gly met gly val ser gly pro glu ile pro leu ala met asp arg phe pro tyr val ala leu ser lys thr tyr asn val asp lys his val pro asp ser gly ala thr ala thr ala tyr leu cys gly val lys gly asn phe gln thr ile gly tyr ala his thr val asn arg asn trp tyr ser asp ala asp val pro ala ser ala arg gln glu gly cys gln asp ile ala thr gln leu ile ser asn met asp ile asp val ile leu gly gly gly arg lys tyr met phe arg met gly thr pro asp pro glu tyr pro asp asp tyr ser gln gly gly thr arg leu asp gly lys asn leu val gln glu trp leu ala lys arg gln gly ala arg tyr val trp asn arg thr glu leu met gln ala ser leu asp pro ser val thr his leu met gly leu phe glu pro gly asp met lys tyr glu ile his arg asp ser thr leu asp pro ser his glu ser arg ala tyr arg ala leu thr glu thr ile met phe asp asp arg lys ala tyr thr val leu leu tyr gly asn gly pro gly tyr val leu lys asp gly ala arg pro asp val thr glu ser glu ser gly ser pro glu tyr arg gln gln ser ala val pro leu asp glu glu thr leu val his gly val gln glu gln thr phe ile ala his val met ala ala lys asn leu ile ile phe leu gly asp gly met gly val ser thr pro glu ile pro leu ala met asp arg phe pro tyr val ala leu ser lys thr tyr asn val asp lys his val pro asp ser gly ala thr ala thr ala tyr leu cys gly val lys gly asn phe gln thr ile gly leu ala his thr val asn arg asn trp tyr ser asp ala asp val pro ala ser ala arg gln glu gly cys gln asp ile ala thr gln leu ile ser asn met asp ile asp val ile leu gly gly gly arg lys tyr met phe gly thr arg leu asp gly lys asn leu val gln glu trp leu ala lys arg gln gly ala arg tyr val trp asn arg thr glu leu met gln ala ser leu asp pro ser val thr his leu met gly leu phe glu pro gly asp met lys tyr glu ile his arg asp ser thr leu asp pro ser leu glu ser arg ala tyr arg ala leu thr glu thr ile met phe asp asp leu val thr ala asp his ser his val phe ser phe gly gly tyr pro leu lys asp gly ala arg pro asp val thr glu ser glu ser gly ser pro glu tyr arg gln gln ser ala val pro leu asp glu glu thr his ala gly glu asp val ala val phe ala arg gly pro gln ala his leu met trp leu val thr phe leu leu leu leu asp ser leu his lys ala arg pro glu asp val gly thr ser leu tyr phe val asn asp ser leu his asp asn asp tyr phe cys thr ala glu asn ala ala gly lys ile arg ser pro asn ile arg val lys ala val phe arg glu pro tyr thr val arg val glu asp gln arg ser met arg gly asn val ala val phe trp glu lys asp thr val ser ile ile pro glu asn arg phe phe ile thr tyr his gly gly leu tyr ile ser asp val gln lys glu asp ala leu ser thr tyr arg cys ile thr lys his lys tyr ser gly glu thr arg gln ser asn gly ala arg leu ser val thr asp pro ala glu ser ile pro thr ile leu asp gly phe his ser gln glu val trp ala gly his thr val glu leu pro cys thr ala ser gly tyr pro ile pro ala ser gly thr tyr ile cys glu val thr asn thr phe gly ser ala glu ala leu thr gly ser pro glu phe thr ile arg trp tyr arg asn thr glu leu val leu pro asp glu ala ile ser ile arg gly leu ser asn glu thr leu leu ile thr ser ala gln lys ser his ser gly ala tyr ile ala leu glu asp gly thr pro arg ile val ser ser phe ser glu lys val val asn pro gly glu gln phe ser leu met cys ala ala lys val arg asp gly ser his arg thr asn gln tyr thr met ser asp gly caggaccacc tccccggaga atagggcctc tttatggc atg tgg ctg gta act ttc 116 ctc ctg ctc ctg gac tct tta cac aaa gcc cgc cct gaa gat gtt ggc 164 leu leu leu leu asp ser leu his lys ala arg pro glu asp val gly acc agc ctc tac ttt gta aat gac tcc ttg cag cag gtg acc ttt tcc 212 agc tcc gtg ggg gtg gtg gtg ccc tgc ccg gcc gcg ggc tcc ccc agc 260 gcg gcc ctt cga tgg tac ctg gcc aca ggg gac gac atc tac gac gtg 308 ccg cac atc cgg cac gtc cac gcc aac ggg acg ctg cag ctc tac ccc 356 pro his ile arg his val his ala asn gly thr leu gln leu tyr pro ttc tcc ccc tcc gcc ttc aat agc ttt atc cac gac aat gac tac ttc 404 tgc acc gcg gag aac gct gcc ggc aag atc cgg agc ccc aac atc cgc 452 cys thr ala glu asn ala ala gly lys ile arg ser pro asn ile arg gtc aaa gca gtt ttc agg gaa ccc tac acc gtc cgg gtg gag gat caa 500 val lys ala val phe arg glu pro tyr thr val arg val glu asp gln agg tca atg cgt ggc aac gtg gcc gtc ttc aag tgc ctc atc ccc tct 548 arg ser met arg gly asn val ala val phe lys cys leu ile pro ser tca gtg cag gaa tat gtt agc gtt gta tct tgg gag aaa gac aca gtc 596 tcc atc atc cca gaa aac agg ttt ttt att acc tac cac ggc ggg ctg 644 ser ile ile pro glu asn arg phe phe ile thr tyr his gly gly leu tac atc tct gac gta cag aag gag gac gcc ctc tcc acc tat cgc tgc 692 tyr ile ser asp val gln lys glu asp ala leu ser thr tyr arg cys atc acc aag cac aag tat agc ggg gag acc cgg cag agc aat ggg gca 740 ile thr lys his lys tyr ser gly glu thr arg gln ser asn gly ala cgc ctc tct gtg aca gac cct gct gag tcg atc ccc acc atc ctg gat 788 ggc ttc cac tcc cag gaa gtg tgg gcc ggc cac acc gtg gag ctg ccc 836 gly phe his ser gln glu val trp ala gly his thr val glu leu pro tgc acc gcc tcg ggc tac cct atc ccc gcc atc cgc tgg ctc aag gat 884 cys thr ala ser gly tyr pro ile pro ala ile arg trp leu lys asp ggc cgg ccc ctc ccg gct gac agc cgc tgg acc aag cgc atc aca ggg 932 gly arg pro leu pro ala asp ser arg trp thr lys arg ile thr gly ctg acc atc agc gac ttg cgg acc gag gac agc ggc acc tac att tgt 980 gag gtc acc aac acc ttc ggt tcg gca gag gcc aca ggc atc ctc atg 1028 glu val thr asn thr phe gly ser ala glu ala thr gly ile leu met gtc att gat ccc ctt cat gtg acc ctg aca cca aag aag ctg aag acc 1076 ggc att ggc agc acg gtc atc ctc tcc tgt gcc ctg acg ggc tcc cca 1124 gag ttc acc atc cgc tgg tat cgc aac acg gag ctg gtg ctg cct gac 1172 glu phe thr ile arg trp tyr arg asn thr glu leu val leu pro asp gag gcc atc tcc atc cgt ggg ctc agc aac gag acg ctg ctc atc acc 1220 tcg gcc cag aag agc cat tcc ggg gcc tac cag tgc ttc gct acc cgc 1268 ser ala gln lys ser his ser gly ala tyr gln cys phe ala thr arg aag gcc cag acc gcc cag gac ttt gcc atc att gca ctt gag gat ggc 1316 acg ccc cgc atc gtc tcg tcc ttc agc gag aag gtg gtc aac ccc ggg 1364 thr pro arg ile val ser ser phe ser glu lys val val asn pro gly gag cag ttc tca ctg atg tgt gcg gcc aag ggc gcc ccg ccc ccc acg 1412 glu gln phe ser leu met cys ala ala lys gly ala pro pro pro thr gtc acc tgg gcc ctc gac gat gag ccc atc gtg cgg gat ggc agc cac 1460 val thr trp ala leu asp asp glu pro ile val arg asp gly ser his cgc acc aac cag tac acc atg tcg gac ggc acc 1493 met trp leu val thr phe leu leu leu leu asp ser leu his lys ala arg pro glu asp val gly thr ser leu tyr phe val asn asp ser leu his asp asn asp tyr phe cys thr ala glu asn ala ala gly lys ile arg ser pro asn ile arg val lys ala val phe arg glu pro tyr thr val arg val glu asp gln arg ser met arg gly asn val ala val phe trp glu lys asp thr val ser ile ile pro glu asn arg phe phe ile thr tyr his gly gly leu tyr ile ser asp val gln lys glu asp ala leu ser thr tyr arg cys ile thr lys his lys tyr ser gly glu thr arg gln ser asn gly ala arg leu ser val thr asp pro ala glu ser ile pro thr ile 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asn gly asn leu tyr ile ala asn val glu ser ser asp arg gly asn pro ala asp ile val val gln phe lys asp ile tyr thr met met gly gln asn val thr leu glu cys phe ala leu gly asn pro val pro asp ile arg trp arg lys val leu glu pro met pro thr thr ala glu ile ser thr ser gly ala val leu lys ile phe asn ile gln leu glu asp glu gly leu tyr glu cys glu ala glu asn ile arg gly lys asp lys his gln ala arg ile tyr val gln ala phe pro glu trp val glu his ile asn asp thr glu val asp ile gly ser asp leu tyr trp pro cys val ala thr gly lys pro ile pro thr ile arg trp leu lys asn gly tyr ala tyr his lys gly glu leu arg leu tyr asp val thr phe glu ser lys gly thr glu trp leu val asn ser ser arg ile leu ile trp ile tyr thr cys phe ala glu asn asn arg gly lys ala asn ser thr asn ala asp ile thr val gly glu asn ala thr met gln cys ala ala tyr val ile asp phe asn lys glu ile thr asn ile his tyr gln arg asn phe met leu asp ala asn gly glu leu leu ile arg asn ala gln leu lys his ala gly arg tyr thr cys thr ala gln thr ile val asp cys ala gly ser ala glu ser lys gly asn arg pro pro arg ile thr lys glu ser asp pro glu arg asn pro phe ile ile glu cys glu ala asp gly gln pro glu pro glu tyr ser trp ile lys asn gly lys lys phe asp trp gln ala tyr asp asn arg met leu arg gln pro gly arg gly thr leu val ile thr ile pro lys asp glu asp arg gly his tyr thr leu glu ala val glu gly glu pro phe met leu lys cys ala ala pro asp gly phe pro ser pro thr val asn trp met ile gln glu ser pro glu gly asn leu trp phe ser asn val thr arg glu asp ala ser arg gln ser ala leu arg gly lys arg met glu leu phe cys ile tyr gly gly thr pro leu pro gln thr val trp ser lys asp gly gln arg ile gln trp ser asp arg ile thr gln gly his tyr gly lys ser leu val ile arg gln thr asn phe asp asp ala gly thr tyr thr cys asp val asn ser val pro tyr phe thr lys glu pro glu ile ala thr ala ser leu gly tyr val tyr lys asp val tyr leu asn val gln ala glu arg asn val thr ile lys cys arg val asn gly ser pro lys pro leu val lys trp leu arg ala ser asn trp leu thr gly gly arg tyr asn val gln ala asn gly asp leu glu ile gln asp val thr phe ser asp ala gly lys tyr thr cys tyr ala gln asn lys phe gly glu ile gln ala asp gly ser leu val val lys glu his thr ile thr gln glu pro gln asn tyr glu val ala ala gly gln ser ala thr phe arg cys asn gly gln ser ile asp phe glu ala gln pro arg phe val lys thr asn asp asn ser leu thr ile ala lys thr met glu leu asp ser gly glu cys leu leu ile gln ile pro glu glu tyr glu gly his his val met pro thr asp asp ile ser leu lys cys glu ala ser gly lys pro glu val gln phe arg trp thr arg asp gly val his phe lys pro lys glu glu leu gly val thr val tyr gln ser pro his ser gly ser phe thr arg cys phe ala ser asn lys leu gly thr ala met ser his glu ile arg leu met ala glu gly ala pro lys trp pro lys glu thr val lys pro pro ser ala glu pro leu arg ile tyr trp met asn ser lys ile leu his ile lys gln asp glu arg val thr met gly gln asn gly asn leu tyr phe ala asn val leu thr ser asp asn his ser asp tyr ile cys his ala his phe pro gly thr arg thr ile ile gln lys glu pro ile asp leu arg val lys ala thr asn ser met ile asp arg lys pro arg leu leu phe pro thr asn ser ser ser his leu val ala leu gln gly gln pro leu val leu glu cys ile ala glu gly phe pro thr pro thr ile lys trp leu arg pro ser gly pro met pro ala asp arg val gln arg gly ala leu ile leu ser asn val gln pro ser asp thr met val thr gln cys glu ala arg asn arg his gly leu leu leu ala asn asn gln thr tyr met ala val pro tyr trp leu his lys pro gln ser his leu tyr gly pro gly glu thr ala arg leu asp cys gln val gln glu glu leu ala lys asp gln gln gly ser thr ala tyr leu leu cys lys ala phe gly ala pro val pro ser val gln trp leu asp glu asp gly thr thr val leu gln asp glu arg phe phe pro tyr ala asn gly thr leu gly ile arg asp leu gln ala asn asp thr gly arg tyr phe lys val lys asp ala thr gln ile thr gln gly pro arg ser thr ile glu lys lys gly ser arg val thr phe thr cys gln ala ser phe asp gln glu leu gly asp ser asp lys tyr phe ile glu asp gly arg leu val ile his ser leu asp tyr ser asp gln gly asn tyr ser cys val phe leu cys gln met ile ser ala leu asp val pro leu asp ser lys lys asp tyr ile val asp pro arg glu asn ile val ile gln cys glu his phe asp ile asp lys asp ala gln val thr met lys pro asn ser gly thr leu val val asn ile met asn gly val lys ala glu ala tyr glu gly val tyr gln cys thr ala arg asn glu arg gly ala ala ile glu lys leu glu pro asn his val arg glu gly asp ser leu val leu asn cys arg pro pro val gly leu pro pro pro ile ile phe trp met asp asn ala phe gln arg leu pro gln ser glu arg val ser gln gly leu asn gly asp leu tyr phe ser asn val gln pro glu asp thr arg val asp tyr ile cys tyr ala arg phe asn his thr gln thr ile gln glu arg pro pro val leu leu thr pro met gly ser thr ser asn lys asp val ser glu ala asp ser gly asn tyr lys cys thr ala arg asn pro tyr trp ile thr ala pro arg asn leu val leu ser pro gly glu asp gly thr leu ile cys arg ala asn gly asn pro lys pro ser ile ser trp leu thr asn gly val pro ile ala ile ala pro glu asp pro ser arg lys val asp gly asp thr ile ile phe ser ala val gln glu leu thr pro ala asn lys leu tyr gln val ile ala asp ser pro ala leu ile asp cys ala tyr phe gly ser pro lys pro glu ile glu trp phe arg gly val lys gly ser ile leu arg gly asn glu tyr val phe his asp asn gly thr leu glu ile pro val ala gln lys asp ser thr gly thr tyr thr cys val ala arg asn lys leu gly lys thr gln asn gln tyr lys val ile gln arg ser ala gln ala ser phe glu cys val leu thr ile met asn val thr asp lys asp asp gly thr tyr thr cys ala ser pro pro ala thr tyr arg trp lys met asn gly thr glu met lys leu glu pro gly ser arg his gln leu val gly gly asn leu val ile met asn pro thr lys ala gln asp ala gly val tyr gln cys leu ala ser asn pro val gly thr val val ser arg glu ala ile leu arg phe gly phe leu gln glu phe ser lys glu glu arg asp pro val lys ala his glu gly trp gly val met leu pro cys asn pro pro ala his ile pro thr asp gly arg his phe val ser gln thr thr gly asn leu tyr ile ala arg thr asn ala ser asp leu gly asn tyr ser cys leu ala gln leu asn leu ala ala glu asp thr arg leu phe ala pro ser ile lys ala arg phe pro ala glu thr tyr ala leu val gly gln gln val thr leu glu cys phe ala phe gly asn pro val pro arg ile lys trp arg lys val asp gly ser leu ser pro gln trp thr thr ala glu pro thr leu gln ile pro ser val ser phe glu asp glu gly thr tyr glu cys glu ala glu asn ser lys gly arg asp thr val gln gly arg ile ile val gln ala gln pro glu trp leu lys val ile ser asp thr glu ala asp ile gly ser asn leu arg trp gly cys ala ala ala gly lys pro arg pro thr val arg trp leu arg asn gly glu pro leu ala ser gln asn arg val glu val leu ala gly asp leu arg phe ser lys leu ser leu glu asp ser gly met tyr gln cys val ala glu asn lys his gly thr ile tyr ala ser ala glu leu ala val gln ala leu ala val val leu trp ser lys gly thr glu ile leu val asn ser ser arg ser asp glu gly lys tyr thr cys phe ala glu asn phe met gly lys ala asn ser thr gly ile leu ser val arg asp ala thr lys ile thr gln cys his ala ser his asp pro thr met asp leu thr phe thr trp ala gln leu arg his gly gly lys tyr thr cys met ala gln thr val val asp ser ala ser lys glu ala thr val leu val arg gly pro asn ser ile phe pro val gly ser glu asp lys lys ile thr leu asn cys glu ala arg gly asn pro ser pro his tyr arg trp gln leu asn gly ser asp ile asp thr ser leu asp his arg tyr lys leu asn gly gly asn leu ile val ile asn pro asn arg asn trp asp thr gly ser tyr gln cys phe ala thr asn ser leu gly thr ile val ser arg glu ala lys leu gln phe ala tyr leu glu asn phe lys ser arg met arg pro pro pro his ser gly glu leu ser tyr ala trp val phe asn glu thr gly his leu tyr ile ala lys val glu pro ser asp val gly asn ser pro thr pro leu val leu arg ser asp gly val met gly glu tyr gly ser thr val lys leu glu cys phe ala leu gly asn pro val pro gln ile asn trp arg arg ser asp gly met pro phe pro thr lys ile lys leu arg lys phe asn gly val leu glu ile pro asn phe gln gln glu asp thr gly ser tyr glu cys ile ala glu asn ser arg gly lys asn val ala arg gly arg leu thr tyr tyr ala lys pro tyr trp val gln leu leu lys asp val glu thr ala val glu asp ser leu tyr trp glu cys arg ala ser gly lys pro lys pro ser tyr arg trp leu lys ala leu thr ile ala asn leu asn val ser asp ser gly met phe gln cys ile ala glu asn lys his gly leu ile tyr ser ser ala glu leu lys val leu ala ser ala pro asp phe ser arg asn pro met lys lys met ile gln val gln val gly ser leu val ile leu asp cys lys pro ser ala ser pro arg ala leu ser phe trp lys lys gly asp thr val val arg glu gln ala arg ile ser leu leu asn asp gly gly leu lys ile met asn val thr lys ala asp ala gly ile tyr thr cys ile ala glu pro thr arg ile ile leu ala pro ser asn met asp val ala val gly glu ser ile ile leu pro cys gln val gln his asp pro leu leu asp ile met phe ala trp tyr phe asn gly thr leu thr asp phe lys leu met ile arg asn ile gln leu lys his ser gly lys tyr val cys met val gln thr gly val asp ser val ser ser ala ala glu leu ile met val leu his ser his gln leu thr tyr ala gly ile ala phe ala val lys asp tyr ile val asp pro arg asp asn ile phe ile glu cys glu ala lys gly asn pro val pro thr phe ser trp thr arg asn gly ser gly thr leu val ile asp phe his gly gly gly arg pro asp asp tyr glu gly glu tyr gln cys phe ala arg asn asp tyr gly thr ala met ser ser ser met glu pro ile his gln asp lys arg val ser gln gly gln asn gly asp leu tyr phe ser asn val met leu gln asp ala gln thr asp tyr ser cys asn ala arg phe his phe thr his thr ile asn glu thr ser leu arg asn his thr asp met tyr ser ala arg gly ser gln met val leu arg gly val asp leu leu leu glu cys ile ala ser gly val pro ala pro asp ile met trp tyr lys lys gly gly glu ile ser asn val ser glu glu asp ser gly glu tyr phe cys leu ala gly glu asp gly arg leu val cys arg ala asn gly asn pro lys pro asn pro ser arg glu val ala gly asp thr ile val phe arg asp thr gln ile gly ser ser ala val tyr gln cys asn ala ser asn glu his arg trp phe lys asn gly gln gly asn met leu asp gly gly asn tyr asp gln gly ile tyr thr cys val ala thr asn ile leu gly lys val glu ala gln val arg leu glu val lys asp pro thr arg ile val arg gly pro glu asp gln val val lys arg gly ser met pro arg leu his lys asp asp ala pro leu tyr ile gly asn arg met lys lys glu asp asp gly leu thr ile tyr gly val ala glu lys asp gln gly asp tyr