Patent Application: US-29418994-A

Abstract:
methods and compositions are provided for cloning and expression of serum opacity factor of streptococcus pyogenes genes . the portion produced by the recombinant dna techniques described herein may be employed in qualitative and quantitative testing for high density lipoprotein , as a fibronectin binding factor and for the regulation of high density lipoprotein in a mammal . the gene may further be employed as a molecular probe for accurate identification of opacity factors from various strains of streptococcus pyogenes .

Description:
fig1 which is not to scale is a sketch showing the general structure of of proteins first isolated and made available in useful highly purified form in accordance with this invention . the skilled artisan will recognize the similarity in structure between this surface protein and the antiphagocytic m protein of streptococcus pyogenes . as shown , and more fully explained below , it is a protein of 1025 residues expressed by a gene having 3107 base pairs with a molecular weight of about 112 , 000 . the protein has a leader sequence at the amino end which is released as the protein becomes bound to the streptococcal surface . the amino end as shown below is the enzyme active segment of the molecule . while there is serological variation among sof from different strains , sufficient homology amongst enzyme active segments from different strains exists so that it is possible to utilize the gene , or gene segment , which expresses the enzyme active segment of one strain as a probe to locate the gene which expresses the enzyme active segment of another strain . each of therefore has at least one apolipoproteinase segment located at amino end of the molecule . the of also includes the hexameric lpxtgx ( seq id no : 1 ) motif characteristic of gram positive bacteria as well as 4 repeats flanked by proline rich stretches within its c - terminal domain . deletion analysis , as shown below , has been used to establish that the c - terminal domain is not involved in the apolipoproteinase activity of the protein . the repeats are the site of the fibronectin binding activity . the protein shown in fig1 is representative of the class of of proteins each having apolipoproteinase activity and isolatable from various streptococcal species . although there is significant homology amongst the proteins from the various strains , there is also appreciable variation . there follows a description of the production and isolation of the dna sequence which expresses the of of the streptococcal strain d734 . the of protein expressed in this representative example is identified as sof22 . the gene used to express the protein is sof22 . this is in accordance with the standard nomenclature system used in this art . related testing , cloning , subcloning , expression and probing procedures are also described . bacterial strains , plasmids and bacteriophages . group a streptococcal strain d734 ( m type 22 ), was the original parent strain of the gene , sof22 , described in this study . this strain and all other group a streptococcal strains used in this study were from the rockefeller university collection and are described in table 1 . e . coli strain p2392 served as the host for lambda phage embl1 . xl1 - blue ( stratagene cloning systems , la jolla , calif .) was the host strain for m13 mp18 / 19 , and plasmid puc9 . 2 and pbluescript sk + . these two e . coli strains did not create an opacity reaction when incubated with heat - inactivated horse serum . the strains and the lambda phage are known and readily available . table 1______________________________________dna hybridization with mpsof89 . 2 probe . m - strain type class hybrid bands sor______________________________________d734 22 ii + 1 . 60 + b234 22 + 3 . 50 , 0 . 60 + b401 22 + 3 . 50 , 0 . 60 + f312 22 + 3 . 50 , 0 . 60 + b344 2 + 1 . 70 + b512 4 + 1 . 60 , 0 . 35 + d691 11 + 1 . 20 , 0 . 23 , 0 . 20 + d474 13 + 1 . 60 + d742 13 + 2 . 50 , 0 . 90 + b737 49 + 0 . 40 + d976 51 + 0 . 80 , 0 . 60 , 0 . 23 + d398 60 + 0 . 18 + a956 62 + 0 . 22 + d459 63 + 0 . 22 + d794 66 + 0 . 23 + d710 1 i - - b788 5 - - d471 6 - - s43 6 + 0 . 80 , 0 . 60 , 0 . 23 - a374 12 + 1 . 90 - d469 12 - - 1rp284 24 - - d617 30 - - d466 37 - - d421 41 + 1 . 90 , 0 . 23 - d463 41 - - d432 54 - - d442 55 - - d735 nt . sup . a - - d739 nt . sup . a - - ______________________________________ chemicals and enzymes . restriction and t4 dna ligase were purchased from new england biolabs , inc . ( beverly , mass .). alkaline phosphatase ( from calf intestine ), human fibronectin and random primed dna labeling kits were obtained from boehringer mannheim corp . ( indianapolis , ind .). dna sequences were determined by using sequenase version 2 . 0 kits ( united states biochemical corp . cleveland , ohio .). unidirectional deletions of m13mp18 / 19 clones were generated using the cyclone 1 biosystem ( international biotechnologies , inc , new haven , conn .). na 125 i and radionucleotides - 32 p ! datp and - 35 s ! datp were obtained from new england nuclear ( boston , mass .). dna oligomers were purchased from operon technologies ( alameda , calif . ), or united states biochemical corp . polymerase chain reactions ( pcr ) were achieved using the geneamp pcr reagent kit ( the perkin - elmer corp ., norwalk , conn .). heat - inactivated horse serum was purchased from life technologies , inc . ( gaithersburg , md .). all other chemicals were purchased from sigma chemical co . ( st . louis , mo . ), unless otherwise indicated . of expressed by colonies of strain d734 was detected by growing bacteria on serum opacity reaction ( sor ) assay medium , containing 50 % heat - inactivated horse serum and todd hewitt broth ( difco laboratories , detroit , mich .) in 0 . 9 % oxoid ion agar no . 2 ( colab laboratories , chicago heights , ill .). recombinant phage plaques expressing of activity were screened on plates containing luria broth instead of todd hewitt broth . after transferring phage lawns to nitrocellulose filters , the of - positive plaques were detected by replica plating the filters on sor assay medium for 10 - 12 h at 37 ° c . serum opacity activity in solutions , including supernatants of phage or bacterial cultures , was measured as previously described . briefly , the liquid solution to be tested was mixed 1 : 5 in heat - inactivited horse serum , containing 0 . 02 % merthiolate , and incubated for 2 h at 37 ° c . opacity was quantitated by spectrophotometric measurement at 475 nm . to visualize of protein bands after denaturing sds - page electrophoresis , the gel ( 10 %) was incubated on solid sor assay medium containing 0 . 02 % merthiolate . of protein was detected as opaque bands in the sor assay medium and photographed under indirect light . late log phase cultures of strain d734 were washed with 200 mm sodium phosphate buffer , ph 7 . 5 , at 4 ° c . and resuspended in 1 / 100 of the original culture volume of the same buffer . extraction of of proceeded by incubating the bacterial suspension of 2 hours at 37 ° c ., followed by centrifugation and filtration of the resulting supernatant through a 0 . 45 um nitrocellulose filter ( schleicher and schuell , inc ., keene , n . h .). of was precipitated from the supernatant in 60 % saturated ammonium sulfate and collected by centrifugation ( 31 , 500 g × 10 min .). the precipitate was resuspended in 100 mm tris - hcl , ph 7 . 5 , and dialyzed in 4 liters of the same buffer for 48 hours at 4 ° c . streptococcus chromosomal dna for all work was prepared by the known phage lysin extraction procedure . chromosomal dna from strain d734 , used in creating recombinant libraries , was either partially digested with sau3ai , or completely digested with ecori . sau3ai digest was dephosphorylated with calf intestinal phosphatase , and ligated to lambda phage embl4 arms digested with bamhi and sa1i . the ecori digest was ligated to dephosphorylated embl4 arms prepared by bamhi and ecori cleavage . ligation mixtures were packaged in vitro using gigapack ii gold packaging extracts ( stratagene ). unamplified libraries were plated on strain p2392 and screened for recombinant phage plaques expressing an of phenotype . four positive phage ( lsof22 . 1 - lsof22 . 3 and lsof22 . 4 from the sau3a and ecori libraries , respectively ) were isolated and plaque purified . for of expression studies and dna sequencing of the 5 &# 39 ; portion of sof22 , a 2 , 560 bp ecori - sa1i fragment , containing the entire 2 , 543 bp insert of phage lsof22 . 3 , was electrophoretioally purified and than ligated to both m13 mp18 and mp19 digested with both ecori and sa1i , creating mpsof22 . 2 and mpsof22 . 1 , respectively . ligation mixtures used to transform xl1 - blue yielded numerous recombinant plaques , as detected by plating on sor assay agar . sets of nested deletion clones for dna sequencing were then prepared from both m13 clones using the cyclone i kit ( ibi ). to sequence the 3 &# 39 ; portion of sof22 , an 3 , 100 bp saci - ecori ( fig3 ) fragment was subcloned from lsof22 . 4 into both m13 mp18 and mp19 , creating mpsof22 . 4 and mpsof22 . 3 , respectively . the 2543 bp insert of phage lsof22 . 3 was also cloned into pbluescript ks + ( psof22 . 1 ). single stranded templates of m13 and mp18 / 19 clones were prepared for chain termination sequencing by known methods . dna sequences of mpsof22 . 1 and mpsof22 . 2 derived deletion clones were determined using the m13 - 20 forward universal primer , sequenase 2 . 0 , and - 35 p ! datp . the 3 &# 39 ; portion of sof22 , cloned in mpsof22 . 3 and mpsof22 . 4 , was sequenced by primer walking . dna sequence data was aligned using the staden program package ( roger staden , mrc laboratory of molecular biology , cambridge , u . k .). this software package was also used to predict structural features of the deduced of protein . the genbank database was accessed to establish regions of homology between sof22 and other known proteins . the rf form of mpsof22 . 3 served as the template for amplification of the fnbd of sof22 . the 5 &# 39 ; primer ,( seq id no : 23 ) cccaagcttcaggaaaataaagat , designed to place the fnbd coding sequence in frame with the upstream lacz gene fragment , corresponded to bases 2641 to 2658 ( fig4 ) and a hindiii site ( underlined ), while the 3 &# 39 ; primer , ( seq id no : 23 ) cgggatccgctcgttatcaaagtgg , consisted of nucleotides 3002 to 2986 ( fig4 ) and a bamhi site . the pcr reaction consisted of 20 cyles of a three step reaction ( 1 min at 94 ° c ., 3 min at 55 ° c ., and 3 min at 72 ° c . ), employing the geneamp pcr reagent kit , native taqdna polymerase , and a dna thermal cycler ( the perkin - elmer corp .). the products , of expected size , from 3 independent pcrs , were pooled , purified by phenol / chloroform extraction , digested with hindiii and bamhi , and isolated from a low melting temperature agarose gel . purified dna was then ligated to the hindiii and bamhi sites of puc9 . 2 , creating the fnbd expressing clone , pfnbd22 . 1 . the recombinant fibronectin binding domain was prepared from the whole cell lysates of clone pfnbd22 . 1 , separated by denaturing sds - page and electroblotted to nitrocellulose . the blots were then blocked by incubation in 10 mm hepes buffer , containing 150 mm naci , 10 10 mm mgcl 2 , 2 mm cacl 2 , 50 mm kcl , 0 . 5 % tween - 20 , 0 . 04 % nan 3 , and 0 . 5 % bsa , ph 7 . 4 , for 2 - 3 h at room temperature . subsequently , blots were then probed for 3 - 4 h at room temperature in the same buffer containing 125 i fibronectin adjusted to 3 × 10 5 cpm / ml . blots were then washed three times with blocking buffer , dried and exposed to kodak blue brand film in the presence of an intensifying screen , for 24 - 36 h , at - 70 ° c . radioiodination of fibronectin was achieved using iodobeads ( pierce chemical ci ., rockford , ill .). the labeled protein was separated from free iodine by filtration through sephadex g - 25 ( pd - 10 ; pharmacia lkb biotechnology inc .) and collected in 100 mm phosphate buffer saline , ph 6 . 5 . the specific activity of the iodinated fibronectin was × 10 6 cpm / ug . crude lysates of clones lsof22 . 3 or embl4 were mixed with purified human hdl in microtiter plate wells and incubated for 16 h at 37 ° c . in the presence of 0 . 02 % nan 3 - hdl was adjusted to a final apoprotein concentration of 30 ug / 100 lysate . when included in the reaction , aspartic protease inhibitor pepstain a ( 5 ug / ml ) was incubated with lambda lysates for 30 min prior to the addition of hdl . opacity reactions were assessed by observation in indirect light . cleavage of apoai was analyzed by denaturing sds - page observation in indirect light . cleavage of apoai was analyzed by denaturing sds - page ( 15 ) of 10 ul of each reaction . electroblotted filters were probed with alkaline phosphatase - conjugated sheep anti - apoai antibody ( biodesign international , kennebunkport , me .) and developed . chromosomal dna prepared from streptococci was digested to completion with restriction enzymes and electrophoresed in 0 . 6 % or 1 . 05 % agarose gels prior to transfer to hybond membranes ( new england nuclear ). dna probes , consisting of either restriction fragments or pcr products , were isolated from preparative low melting temperature agarose gels and then radiolabeled with 32 p ! datp , using random primed dna labelling kits ( boehringer mannheim corp .). blots were incubated in a prehybridization solution ( 6 × ssc , 0 . 5 % sds , 100 ug / ml denatured salmon sperm dna , 5 × denhardt &# 39 ; s solution and 50 % formamide ), at 30 ° c . overnight , and then hybridized with radiolabeled probes in a hydrization solution ( 6 × ssc , 0 . 5 % sds , 100 ug / ml denatured salmon sperm dna and 50 % formamide ), at 42 ° c . overnight in accordance with known procedures . blots that physically mapped the sof22 and emm2 loci were washed at high stringency conditions that allowed for less than 5 % bp mismatch ( twice in 0 . 1 % ssc and 0 . 1 % sds for 30 min at 65 ° c .). probes used for restriction mapping of sof22 were inserts from m13 deletion clones mpsof22 . 81 and mpsof22 . 792 , corresponding to nucleotides 1 to 781 and 1 , 160 to 2 , 543 , respectively ( fig4 ). blots detecting sof22 homologs in other group a streptococcal strains were sequentially washed at both high and low stringency conditions . whereas high stringency washes allowed for less than 5 % mismatched , relaxed wash conditions ( twice in 0 . 2 % ssc and 0 . 5 % sds for 30 min at 37 ° c .) allowed up to 20 % mismatch . the probe in these experiments was the insert from the m13 clone mpsof22 . 692 , corresponding to an internal sau3a fragment ( bp 976 to 2 , 543 ) encoding the serum opacity domain ( sod ). the serum opacity factor of group a streptococci can be detected in both extracellular and membrane bound fractions isolated from bacterial cell cultures . to both ascribe the basis of the serum opacity reaction to a specific molecule ( s ) and judge the feasibility of molecularly cloning the serum opacity factor , a maximum yield method to extracting the released form of the factor was established as described above . the protein extract of sof - producing strain d734 was analyzed by sds - page under denaturing conditions and used in a serum opacity reaction assay . as can be seen in fig2 these methods isolated and detected at least two closely migrating species , with molecular weights of 100 kd that are capable of independently producing a serum opacity reaction . thus , a simple and dependable assay capable of detecting molecules responsible for a serum opacity reaction was established . two streptococcus genomic libraries , containing either sau3ai or ecori restriction fragments of strain d734 chromosomal dna , were constructed in embl4 vector and screened for recombinant phage clones exhibiting a serum opacity phenotype . for the detection of sof protein expression in phage plaques , the method described above was employed . by assaying for positive plaques in this manner , four phages , sof22 . 1 to sof22 . 3 from the sau3ai library and sof22 . 4 from the ecori library , were identified , isolated , and confirmed as serum opacity producing phages . crude phage lysates of clones sof22 . 1 to sof22 . 3 were all found to contain a protein with serum opacity activity that corresponds in size , 100 kda , with the streptococcal serum opacity factor ( fig2 ). in contrast , phage sof22 . 4 expressed at least four different molecular species , ranging from 100 kda to 175 kda in size , with serum opacity activity ( fig2 ). the differences in both the number and mobility of the major reactive species of sof expressed by recombinant clones sof22 . 1 to sof22 . 3 and sof22 . 4 were found to parallel the insert sizes of these clones . all three clones from the sau3ai library had identical 2 . 5 kb inserts flanked by embl4 linkers and saii stuffer fragments . phage sof22 . 4 harbored an ecori fragment , which contained the sau3ai cloned in lsof22 . 1 - lsof22 . 3 ( fig3 ). thus , with codon requirements for the expression of a 100 kd protein in mind ( typically 3 kb ), it was concluded that phages sof22 . 1 to sof22 . 3 encode a truncated sof22 gene , and that , minimally , phage sof22 . 4 encodes the entire sof22 locus . in that one species of sof released from streptococci comigrated with the solitary sof species expressed by sof22 . 3 , we concluded that at least one form of sof released from streptococci is smaller than the native cell bound form . these were verified by sequencing subclones of these phages ( described below ). to prepare dna templates for dna sequencing , the sof22 . 3 phage insert was sublconed in m13mp18 and mp19 , creating mpsof22 . 2 and mpsof22 . 1 , respectively . the insert was , therefore , in both orientations relative to the lacz promoter . sof was expressed from both recombinant phages . increased levels of iptg did not increase the level of periplasmic sof activity . thus , it was indicated that sof22 was expressed from its streptococcal promoter in these recombinant phages . sequence analysis of the 2 , 543 bp fragment from a set of nested deletion clones derived from mpsof22 . 2 and mpsof22 . 1 detected an orf , 2 , 470 nucleotides long , starting at nucleotide 83 and lacking the stop codon . all other orfs detected were smaller than 1 , 500 pb . the 2 , 470 bp long orf , ( sof22 ), was the only one corresponding to the size of sof22 produced by lsof22 . 3 . the 3 &# 39 ; portion of sof22 of orf was located on a 3 , 100 bp saci - ecori fragment by hybridization , using a probe homologous to the sequences downstream from a unique saci 941 site ( fig3 ). this 3 , 100 bp saci - ecori restriction fragment , contained in the ecori insert of phage sof22 . 4 , was subcloned in both m13mp18 and mp 19 , and sequenced by primer walking . the 3 &# 39 ; end of sof22 orf was located at nucleotide 3 , 178 , 644 bp downstream of the end of 2 , 543 sau3a fragment . the nucleotide sequence of sof22 with its deduced amino acid sequence is shown in fig4 . the nucleotide sequence begins at the sau3a site ( position 1 ) and ends at position 3 , 240 . restriction sites xhoi , saci , psti and one sau3a ( the 3 &# 39 ; end of sau3a lsof 22 . 3 insert ) are indicated in the figure . sof22 orf starts at the position 83 and ends at the position 3 , 187 . the longest protein that could be coded for by this orf starts with the alternate start codon uug at position 113 and codes for a protein of 1 , 025 aminoacids with a size 112 , 735 . 1 , about 10 % larger than the largest major sof band released from d734 . although there were a few standard ( aug ) start codons downstream of proposed uug codon , none of the deduced protein sequences contained hydrophobic signal sequence that would allow export of sof from the cytoplasm ( fig5 c ). position of the signal sequence cleavage site , predicted by the method of von heijne , was between g 29 and q 30 . analysis of the amino acid composition of of22 protein revealed that the most abundant amino acids were lysine ( 10 . 54 %), serine ( 9 . 27 %), threonine ( 8 . 29 %) and glutamine ( 8 . 00 %). secondary structure analysis predicts a protein consisting of 39 . 3 % helix , 28 . 2 % random coils , 18 . 15 % beta sheet and 14 . 3 % turns ( fig5 b ). consistant with the low helix potential for the molecule , analysis of the sequence employing the &# 34 ; matcher &# 34 ; algorithm showed no significant seven residue periodicity , excluding the possibility of coiled coil structure . hydrophaticity analysis by the kyte and doolittle algorithm shows strong hydrophobic regions both at the first n - terminal 30 amino acids , in the position of the signal sequence , and at the c terminus , in the position of a possible membrane anchor segment ( fig5 c ). c - terminal to the hydrophobic segment ( at the c - terminal end of the protein ) are eight charged and two polar amino acids . three residues n - terminal from the hydrophobic domain is a hexameric lpxtgx ( seq id no : 1 ) motif found in the surface proteins from gram - positive bacteria , with conservative replacement t to s . analysis of internal homology by diagon proportional algorithm showed 4 repeats ( fig7 ). repeats are flanked by proline rich stretches ( fig5 a , 6a ). all these motifs are present in the majority of streptococcal surface proteins . the 214 c - terminal amino acids , including repeats and downstream proline - rich region , are dispensable for serum opacity activity , as concluded from the sof activity of the truncated sof22 . further shortening of 2 , 543 bp sau3a fragment in clone mpsof22 . 1 by the deletion of 60 c - terminal amino acids ( 180 3 &# 39 ; base pairs ) abolishes sof activity ( fig2 ). therefore , the sequence necessary sof activity is between amino acid 752 and 811 . sequence homology with the membrane anchor region of streptococcal surface proteins comparisons of deduced amino acid sequence of the sof22 protein with a protein sequence database revealed homologies of about 30 to 35 % confined primarily to the c terminal region , including the lpxtgx ( seq id no : 1 ) motif , hydrophobic domain and charged tail of streptococcal proteins deduced from dna sequence . these include ennx and emm49 genes from m type 49 , emm6 from m type 6 , emm12 from m type 12 , emm5 from m type 5 , protein g protein h and protein arp4 . comparison of the anchor region of several surface proteins from the gram positive bacteria is shown in fig7 . putative promoter region shows significant homology to the scpa promoter . comparison of the two sequences shown on fig9 yields highest degree of homology in the regions around - 10 box and upstream of - 35 box including 2 putative virr binding consensus elements upstream of - 35 box . probe mpsof69 . 2 homologous to 1 . 5 kb long internal sau3a fragment ( nucleotide 890 to 2465 ) was used for the southern blot analysis of chromosomal dna sau3a digests of 15 of + and 15 of - strains that belong to different m types ( fig9 table 1 ). this probe detects the sequence encoding for the functional domain of of . under high stringency washing conditions only dna of strain d734 , from which the sof22 gene is cloned , hybridized with the probe . under relaxed stringency washing ( allows 20 % mismatch ) hybridization patterns obtained with dnas from non - m22 sof producing strains were different from that of d734 . unexpectedly , the signals obtained from the strains of the same m types differed among themselves . both the intensity of the signal and the restriction fragment pattern ( length and number ) were different for the two strains of m type 13 . two different patterns were obtained within the m - type 22 strains . 4 m22 strains analyzed fall into the two groups : b243 , b401 and f312 in one , and d734 in the other . out of 15 of - strains analyzed one of the 2 m6 strains and a m12 strain gave positive signals . the only class i strain that gave a positive sof reaction was d421 ( m41 ). it gave a positive signal upon southern hydridization ( relaxed conditions ). the other m41 strain tested was of - and showed no signal in the southern blot analysis . two probes homologous to the sequences upstream ( mpsof2279 . 2 ) and downstream ( mpsof228 . 1 ) of the xhoi and saci sites , were used in southern blots to construct a physical map around sof22 gene ( fig3 ). nonoverlapping restriction map was constructed from the autoradiographs obtained after hybridization of an emm22 probe . calculated from the size of the largest restriction fragments on which sof22 is positioned , its distance from the emm22 gene is at least 30 kbs in the upstream and 15 kbs in the downstream direction . in fig4 the of segment is represented by amino acid residues 1 to 1025 . the enzymatic active segment is amino acid 1 to 811 . the segment 1 to 29 is the leader sequence , lsof 22 . 4 and psof22 . 1 identified above have been deposited at the american type culture collection under the numbers atcc 75541 and atcc 75542 . what has been described is a cloning and subcloning procedure in which a dna sequence comprising of from d734 has been isolated and subcloned to identify the enzyme active segment . the of of d734 and the enzyme active segment are useful to qualitatively and quantitatively identify hdl by contact with a mammalian fluid suspected of containing it and determining if there has been a reaction . one procedure is to observe the development of opacity qualitatively . the determination can be made quantitative by labeling the protein , for example with a radioactive or enzymatic label and comparing the results with known or preestablished standard curves . the degree of opacity could also be used for quantitation , using a standard curve of known hdl . the insertion of the selected dna sequence into a phage or a plasmid to permit the expression of a selected of or enzyme active segment has also been described . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 23 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( v ) fragment type : c - terminal ( xi ) sequence description : seq id no : 1 : leuproxaathrglyxaa15 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 3240 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( iii ) hypothetical : no ( iv ) anti - sense : no ( ix ) feature :( a ) name / key : - 35_signal ( b ) location : 34 .. 39 ( ix ) feature :( a ) name / key : - 10_signal ( b ) location : 57 .. 62 ( ix ) feature :( a ) name / key : rbs ( b ) location : 104 .. 108 ( ix ) feature :( a ) name / key : cds ( b ) location : 113 .. 3190 ( xi ) sequence description : seq id no : 2 : gatcattaatttttatctcaccaaaaaactgattttagaaacgaaaaagcatggtgtata60ataaagttcggaacaattatgacattataatgaaagtaaggttaacgaaacattg115leuacaaattgtaagtataaacttagaaagttatctgtagggctcgtctcc163thrasncyslystyrlysleuarglysleuservalglyleuvalser51015gtcggaacgatgctgatagccccgacagttttaggacaggaggttaat211valglythrmetleuilealaprothrvalleuglyglngluvalasn202530gctagtactgagacgagtgctagtagtactactagtaccgctagcacc259alaserthrgluthrseralaserserthrthrserthralaserthr354045gctgagactagcactcctaccggtacgagtggaacagctgccagcgga307alagluthrserthrprothrglythrserglythralaalasergly50556065gctagtggtgaagcaaccgtagctactgccaatggaggaccccagtct355alaserglyglualathrvalalathralaasnglyglyproglnser707580gctcctgcaacatctgaagcgactccacaacctcaagcacaggcagct403alaproalathrserglualathrproglnproglnalaglnalaala859095ccagcagcatctgcccccactactgtgacctcttctagttctagtgat451proalaalaseralaprothrthrvalthrserserserserserasp100105110agtgacgcgaaaactcctaaggcagcaagcactacatcatcttcagca499seraspalalysthrprolysalaalaserthrthrserserserala115120125actgtggctagccctagtaatggtagcaataaagaagctaatgctgag547thrvalalaserproserasnglyserasnlysglualaasnalaglu130135140145actgcaccacagatgatggacgtggaacagtataagataaaagatgaa595thralaproglnmetmetaspvalgluglntyrlysilelysaspglu150155160aattcttctattactgttgcagataaagctaaacaattaaagatccga643asnserserilethrvalalaasplysalalysglnleulysilearg165170175cgagatgataatccaaaagacaaggatcttttcgatgtcaaacgtgaa691argaspaspasnprolysasplysaspleupheaspvallysargglu180185190gtaaaagataatggcgatggaaccttagatgtaaccttaaaagtaatg739vallysaspasnglyaspglythrleuaspvalthrleulysvalmet195200205cctaaacaaattgacgaaggtgccgatgttatggcccttttagatgtc787prolysglnileaspgluglyalaaspvalmetalaleuleuaspval210215220225tctcaaaagatgacaaaagagaattttgataaggctaaagaacaaata835serglnlysmetthrlysgluasnpheasplysalalysgluglnile230235240aaaaaaatggttacaactttaacaggcgagccaactgatggtaaggaa883lyslysmetvalthrthrleuthrglygluprothraspglylysglu245250255aatcataataggcgtaattctgtacgtctaatgactttttaccgtaag931asnhisasnargargasnservalargleumetthrphetyrarglys260265270gttagcgatccgattgagctcactacaaaaaacgttgatgctaaatta979valseraspproilegluleuthrthrlysasnvalaspalalysleu275280285aaggaagtttgggatcaggccaaaaaagattgggactggggtgttgat1027lysgluvaltrpaspglnalalyslysasptrpasptrpglyvalasp290295300305ttacaaggcgctatccataaggctcgagaaatttttaagaaagaaaaa1075leuglnglyalailehislysalaarggluilephelyslysglulys310315320aagtcaaaaaaacgccaacatatcgtcctgttctctcaaggcgagtca1123lysserlyslysargglnhisilevalleupheserglnglygluser325330335acctttagttatgacattcataacaaaagtgattccaaaattctaaaa1171thrphesertyraspilehisasnlysseraspserlysileleulys340345350acaagggtaaatgaaaatatcacaacttctaacccactgtttccctgg1219thrargvalasngluasnilethrthrserasnproleupheprotrp355360365cttcccatctttaaccatacgaatcgtaaagcagacatgattgatgat1267leuproilepheasnhisthrasnarglysalaaspmetileaspasp370375380385gttaagtatcttattaagtggggtgaaaaattagggatagaagggcta1315vallystyrleuilelystrpglyglulysleuglyilegluglyleu390395400aatgacctagataatacattaaaattagcaggagcagctagtggaatt1363asnaspleuaspasnthrleulysleualaglyalaalaserglyile405410415gtaggtggttttttaggtggaggtagtctaacggagtatcttagcctt1411valglyglypheleuglyglyglyserleuthrglutyrleuserleu420425430aaagaatatcagtcagacaggcttaatgcaagtcaatttaattatgaa1459lysglutyrglnseraspargleuasnalaserglnpheasntyrglu435440445agacgtgttggtgaagggtattattaccatagtttttctgaaaggaaa1507argargvalglygluglytyrtyrtyrhisserphesergluarglys450455460465actgctgaaatgccgaacagagcacttattaagaaacaattagaaggc1555thralaglumetproasnargalaleuilelyslysglnleuglugly470475480ctatttaagggaaaagaaggtaaatggtttaagtctattttagaaaaa1603leuphelysglylysgluglylystrpphelysserileleuglulys485490495ttatcacttacagatgattatcaaaaagcaaaagaagaagctattttg1651leuserleuthraspasptyrglnlysalalysgluglualaileleu500505510aaagtgcttgattacttcttttacaaaagagactatatttactacaat1699lysvalleuasptyrphephetyrlysargasptyriletyrtyrasn515520525cacaatctctcagcaatagctgaagccaaaatggctcaacaagagggg1747hisasnleuseralailealaglualalysmetalaglnglnglugly530535540545gtcaccttctattccgttgatgttactgatttcaactcagcttctaaa1795valthrphetyrservalaspvalthrasppheasnseralaserlys550555560agagcaaagcgacaagtaaaaagtgaagaggataagaaaaaagcaaaa1843argalalysargglnvallysserglugluasplyslyslysalalys565570575gagaaggagaacattgaaaaaaaacgtgacgaaaagtttgataattac1891glulysgluasnileglulyslysargaspglulyspheaspasntyr580585590ttaaaacaaatgtctgaaggcggtaaagaattttttaacgatgtggat1939leulysglnmetsergluglyglylysgluphepheasnaspvalasp595600605aaggcagagaatttcaaagataccctaaccagtgtgacagtgacagag1987lysalagluasnphelysaspthrleuthrservalthrvalthrglu610615620625acttttgggaacaacgtgtctgttgagagtggttcatggaaaacttca2035thrpheglyasnasnvalservalgluserglysertrplysthrser630635640ctaggtagtaatagtggttcaagtagcagagaggtttcctataaagga2083leuglyserasnserglyserserserarggluvalsertyrlysgly645650655cgggatagtggaagtctattttcacttttcggtagtaccaaagaaagt2131argaspserglyserleupheserleupheglyserthrlysgluser660665670ctcacttggactatttccaaagaccagttgaaacaagcctttgaagag2179leuthrtrpthrileserlysaspglnleulysglnalaphegluglu675680685ggtaagccgctaaccctcacctataagctgaaagttgataaagataaa2227glylysproleuthrleuthrtyrlysleulysvalasplysasplys690695700705tttagagaaactcttaaaaagcaacaagaatctcgtcgtataaagaaa2275phearggluthrleulyslysglnglngluserargargi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( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( v ) fragment type : internal ( xi ) sequence description : seq id no : 3 : leuproalaserglyasp15 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 6 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( v ) fragment type : internal ( xi ) sequence description : seq id no : 4 : serproxaathrglyxaa15 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 120 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : unknown ( ii ) molecule type : peptide ( v ) fragment type : c - terminal ( vi ) original source :( a ) organism : streptococcus pyogenes ( b ) strain : d734 ( c ) individual isolate : 22 ( vii ) immediate source :( b ) clone : sau3a ( xi ) sequence description : seq id no : 5 : glngluasnlysaspproilevalaspilethrgluaspthrglnpro151015glymetserglyserasnaspalathrvalvalglugluaspthrthr202530proglnargproaspvalleuvalglyglyglnseraspproileasp354045ilethrgluaspthrglnprosermetserglyserasnaspalathr505560valvalglugluaspvalthrproglnargproaspileleuvalgly65707580glyglnseraspproileaspilethrgluaspthrglnprosermet859095serglyserasnaspalathrvalileglugluaspthrlysprolys100105110argphephehispheaspasnglu115120 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 39 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : unknown ( ii ) molecule type : peptide ( vi ) original source :( a ) organism : streptococcus pyogenes ( b ) strain : d734 ( c ) individual isolate : 22 ( vii ) immediate source :( b ) clone : sau3a ( xi ) sequence description : seq id no : 6 : valvalglugluaspthrthrproglnargproaspvalleuvalgly151015glyglnseraspproileaspilethrgluaspthrglnprosermet202530serglyserasnaspalathr35 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 37 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : unknown ( ii ) molecule type : peptide ( vi ) original source :( a ) organism : streptococcus pyogenes ( b ) strain : d734 ( c ) individual isolate : 22 ( vii ) immediate source :( b ) clone : sau3a ( xi ) sequence description : seq id no : 7 : valgluthrgluaspthrlysgluproglyvalleumetglyglygln151015sergluservalgluphethrlysaspthrglnthrglymetsergly202530glnthrthrprogln35 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 38 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : unknown ( ii ) molecule type : peptide ( vi ) original source :( a ) organism : streptococcus pyogenes ( b ) strain : d734 ( c ) individual isolate : 22 ( vii ) immediate source :( b ) clone : sau3a ( xi ) sequence description : seq id no : 8 : ileileglugluaspthrasnlysasplysprosertyrglnphegly151015glyhisasnservalasppheglugluaspthrleuprolysvalser202530glyglnasngluglygln35 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 51 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : unknown ( ii ) molecule type : peptide ( v ) fragment type : c - terminal ( vi ) original source :( a ) organism : streptococcus pyogenes ( b ) strain : d734 ( c ) individual isolate : 22 ( vii ) immediate source :( b ) clone : sau3a ( xi ) sequence description : seq id no : 9 : serleuserleuproglnalaprovaltyrlysalaalahishisleu151015proalaserglyasplysargglualaserphethrilevalalaleu202530thrileileglyalaalaglyleuleuserlyslysargargaspthr354045glugluasn50 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 50 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : unknown ( ii ) molecule type : peptide ( iii ) hypothetical : no ( v ) fragment type : c - terminal ( vi ) original source :( a ) organism : streptococcus pyogenes ( xi ) sequence description : seq id no : 10 : lysproasnglnasnlysalaprometlysgluthrlysargglnleu151015proserthrglygluthralaasnprophephethralaalaalaleu202530thrvalmetalathralaglyvalalaalavalvallysarglysglu354045gluasn50 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 50 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : unknown ( ii ) molecule type : peptide ( iii ) hypothetical : no ( v ) fragment type : c - terminal ( xi ) sequence description : seq id no : 11 : lysproasnglnasnlysalaprometlysgluthrlysargglnleu151015proserthrglygluthralaasnprophephethralaalaalaleu202530thrvalmetalathralaglyvalalaalavalvallysarglysglu354045gluasn50 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 49 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : unknown ( ii ) molecule type : peptide ( iii ) hypothetical : no ( v ) fragment type : c - terminal ( vi ) original source :( a ) organism : streptococcus pyogenes ( xi ) sequence description : seq id no : 12 : glnalaasnargserargseralametthrglnglnlysargthrleu151015proserthrglygluthralaasnprophephethralaalaalaala202530thrvalmetvalseralaglymetleualaleulysarglysgluglu354045asn ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 49 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : unknown ( ii ) molecule type : peptide ( iii ) hypothetical : no ( v ) fragment type : c - terminal ( vi ) original source :( a ) organism : streptococcus pyogenes ( xi ) sequence description : seq id no : 13 : glnthralathrargproserglnasnlysglymetargserglnleu151015proserthrglyglualaalaasnprophephethralaalaalaala202530thrvalmetvalseralaglymetleualaleulysarglysgluglu354045asn ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 50 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : unknown ( ii ) molecule type : peptide ( iii ) hypothetical : no ( v ) fragment type : c - terminal ( vi ) original source :( a ) organism : group g streptococci ( xi ) sequence description : seq id no : 14 : lyslysproglualalyslysaspaspalalyslysalagluthrleu151015prothrthrglygluglyserasnprophephethralaalaalaleu202530alavalmetalaglyalaglyalaleualavalalaserlysarglys354045gluasp50 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 47 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : unknown ( ii ) molecule type : peptide ( iii ) hypothetical : no ( v ) fragment type : c - terminal ( vi ) original source :( a ) organism : streptococcus mutans ( xi ) sequence description : seq id no : 15 : thrthrthrserlysglnvalthrlysglnlysalalysphevalleu151015proserthrglygluglnalaglyleuleuleuthrthrvalglyleu202530valilevalalavalalaglyvaltyrphetyrargthrargarg354045 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 50 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : unknown ( ii ) molecule type : peptide ( iii ) hypothetical : no ( v ) fragment type : c - terminal ( vi ) original source :( a ) organism : staphylococcus aureus ( xi ) sequence description : seq id no : 16 : lyslysglnproalaasnhisalaaspalaasnlysalaglnalaleu151015progluthrglyglugluasnproleuileglythrthrvalphegly202530glyleuserleualaleuglyalaalaleuleualaglyargargarg354045gluleu50 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 52 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : unknown ( ii ) molecule type : peptide ( iii ) hypothetical : no ( v ) fragment type : c - terminal ( vi ) original source :( a ) organism : staphylococcus aureus ( xi ) sequence description : seq id no : 17 : lysalavalalaprothrlyslysproglnserlyslyssergluleu151015progluthrglyglyglugluserthrasnlysglymetleuphegly202530glyleupheserileleuglyleualaleuleuargargasnlyslys354045asnhislysala50 ( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 56 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : unknown ( ii ) molecule type : peptide ( iii ) hypothetical : no ( v ) fragment type : c - terminal ( vi ) original source :( a ) organism : streptococcus pyogenes ( xi ) sequence description : seq id no : 18 : serserlysargalaleualathrlysalaserthrargaspglnleu151015prothrthrasnasplysaspthrasnargleuhisleuleulysleu202530valmetthrthrphephepheglyleuvalalahisilephelysthr354045lysargglnlysgluthrlyslys5055 ( 2 ) information for seq id no : 19 :( i ) sequence characteristics :( a ) length : 50 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : unknown ( ii ) molecule type : peptide ( iii ) hypothetical : no ( v ) fragment type : c - terminal ( xi ) sequence description : seq id no : 19 : valglugluasnargglulysprothrlysasnilethrproileleu151015proalathrglyaspaspilegluasnvalleualapheleuglyile202530leuileleuservalleuproilepheserleuleulyslysglnthr354045lysgln50 ( 2 ) information for seq id no : 20 :( i ) sequence characteristics :( a ) length : 69 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( iii ) hypothetical : no ( iv ) anti - sense : no ( vi ) original source :( a ) organism : streptococcus pyogenes ( b ) strain : d734 ( c ) individual isolate : 22 ( vii ) immediate source :( b ) clone : sau3a ( xi ) sequence description : seq id no : 20 : gatcattaatttttatctcaccaaaaaactgattttagaaacgaaaaagcatggtgtata60ataaagttc69 ( 2 ) information for seq id no : 21 :( i ) sequence characteristics :( a ) length : 71 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( iii ) hypothetical : no ( iv ) anti - sense : no ( xi ) sequence description : seq id no : 21 : aggtcacaaactaaacaactcttaaaaagctgacctttactaataatcgtctttttttta60taataaagatg71 ( 2 ) information for seq id no : 22 :( i ) sequence characteristics :( a ) length : 24 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( iii ) hypothetical : no ( iv ) anti - sense : no ( xi ) sequence description : seq id no : 22 : cccaagcttcaggaaaataaagat24 ( 2 ) information for seq id no : 23 :( i ) sequence characteristics :( a ) length : 25 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( iii ) hypothetical : no ( iv ) anti - sense : no ( xi ) sequence description : seq id no : 23 : cgggatccgctcgttatcaaagtgg25__________________________________________________________________________