Patent Application: US-32185702-A

Abstract:
methods for improving binding of a proteinaceous substance to cell - wall material of a gram - positive bacterium are disclosed . the proteinaceous substance includes an acma cell - wall binding domain , homolog or functional derivative thereof . the method includes treating the cell - wall material with a solution capable of removing a cell - wall component such as a protein , lipoteichoic acid or carbohydrate from the cell - wall material and contacting the proteinaceous substance with the cell - wall material .

Description:
acid pretreatment of gram - positive bacteria enhances binding of acma protein anchor fusions . bacterial strains and growth conditions . lactococcus lactis strain mg1363 ( gasson 1983 ) or derivatives thereof , such as mg1363δacma ( buist et al . 1995 ) or nz9000δacma , were used as recipients for binding of reporter fusion protein . nz9000 ( kuipers et al . 1997 ) which carries one of the reporter plasmids was used as a production strain . l . lactis strains were grown in m17 broth ( oxoid ) supplemented with 0 . 5 % glucose in standing cultures at 30 ° c . chloramphenicol was added to the m17 medium to an end - concentration of 5 μg / ml when appropriate . for expression , mid - log phase cultures were induced for 2 hours with the culture supernatant of the nisin producing l . lactis strain nz9700 as described by kuipers et al . ( 1997 ). lactobacillus casei , atcc393 , was grown in mrs broth ( oxoid ) in standing cultures at 30 ° c . mycobacterium smegmatis , atcc700084 , was grown in middlebrook medium ( oxoid ) at 37 ° c . in aerated cultures . bacillus subtilis , 168 , was grown in ty broth ( per liter : 10 g tryptone , 5 g yeast extract , 5 g nacl ph7 . 4 ) at 37 ° c . in aerated cultures . construction of reporter plasmids . the merozoite surface antigen 2 ( msa2 ) of plasmodium falciparum strain 3d7 ( ramasamy et al . 1999 ) fused to the three repeats of acma ( msa2 :: ca ) was used as the reporter anchor protein . the reporter anchor protein is encoded by plasmid png3041 based on the nisin inducible expression vector pnz8048 ( kuipers et al . 1997 ) and contains a modified multiple cloning site in which the hybrid reporter gene was cloned . an in frame fusion of the reporter was made at the 5 ′ end the lactococcal prtp signal — and prosequence , and at the 3 ′ end the acma protein anchor sequence . the sequence of the msa2 gene that was included in the construct corresponds to nucleotides ( nt ) 61 to 708 in genbank accession number a06129 . primers used for the amplification of the msa2 gene were msa2 . 1 ( 5 ′- accatggcaaaaaatgaaagtaaatatagc ( seq id no : 2 )) and msa2 . 4 ( 5 ′- cggtctctagcttataagcttagaattcgggatgttgctgctcc acag ( seq id no : 3 )). the primers contain tags with restriction endonuclease recognition sites that were used for cloning . for cloning of the prtp signal and prosequence ( nt 1206 to 1766 in kok et al . 1988 ), the primers prtp . sspro . fw ( 5 ′- ccgtctcccatgcaaaggaaaaaaga aagggc ( seq id no : 4 )) and prtp . sspro . rev ( aaaaaaagcttgaattcccat ggcagtcggataataaactttcgcc ( seq id no : 5 )) were used . the primers include restriction sites that were used for cloning . the acma protein anchor gene fragment ( nt 833 to 1875 ) was obtained by subcloning a pvuii - hindiii fragment from plasmid pal01 ( buist et al . 1995 ). restriction endonuclease enzymes and expand high fidelity pcr polymerase were used in accordance with the instructions of the supplier ( roche ). the final expression vector was designated png3041 ( fig1 ). a construct including a stopcodon introduced after the msa2 sequence in png3041 was designated png304 . the protein secreted using this construct is substantially the same as the protein expressed from the png3041 plasmid except that the protein produced from png304 does not contain the acma protein anchor . the protein produced from png304 is used as a negative control in the binding assays . a vector was also made in which the acma protein anchor was exchanged for a protein anchor . the putative cell - wall binding domain of l . lactis acmd ( bolotin et al . 2001 ) was cloned ( nt 1796 to 2371 in genbank accesssion number ae006288 ) using primers pacmb2 ( 5 ′- cgcaagcttctgcagagctcttagattctaatt gtttgtcctgg ( seq id no : 6 )) and pacmb3 ( 5 ′- cggaattcaaggaggagaaata tcaggagg ( seq id no : 7 )) to produce the plasmid png3042 . png3042 contains an in - frame fusion between msa2 and the protein anchor of acmd ( msa2 :: cd ) and differs from plasmid png3041 only in the gene fragment encoding the protein anchor . cell pretreatment and binding conditions . chemical pretreatment of l . lactis nz9000äacma was done with 10 % tca ( 0 . 6 m ) in the following manner . cells of 0 . 5 ml stationary phase cultures were sedimented by centrifugation and washed once with 2 volumes demineralized water . cells were resuspended in 1 volume of a 10 % tca solution and incubated by placing the reaction tube in boiling water for 15 minutes . subsequently , cells were washed once with 2 volumes pbs ( 58 mm na 2 . hpo 4 . 2h 2 o , 17 mm nah 2 po 4 . h 2 o , 68 mm nacl ; ph 7 . 2 ) and three times with 2 volumes demineralized water . the cells were used directly for binding experiments or stored ( as described herein ) until further use . the following chemicals and conditions were used to examine the effect of different chemicals on the binding capacity of l . lactis cells for acma - type protein anchor fusions : acetic acid ( hac ), hydrochloric acid ( hcl ), sulfuric acid ( h 2 so 4 ), tca , and trifluoroacetic acid ( tfa ), monochloro acetic acid ( mca ). the acids were used at a final concentration of 0 . 6 m and incubated for 15 minutes in boiling water . sds , dimethyl formamide ( dmf ) and dimethyl sulfoxide ( dmso ) were used at a concentration of 10 %. the sds pretreatment was incubated for 15 minutes in boiling water and dmf and dmso treatments were incubated at room temperature for 15 minutes . cells were also pretreated with phenol ( tris buffer saturated ) and incubated for 15 minutes at 55 ° c . other chemicals pretreated at the 55 ° c . incubation temperature were : 4 m guanidine hydrochloride ( gnhcl ), 37 % formaldehyde , chloroform : methanol ( chcl 3 : ch 3 0h ( 2 : 1 )) and 0 . 1 % sodium , hypochlorite ( naocl ). in addition , incubation with 25 mm dithiothrietol ( dtt ) for 30 minutes at 37 ° c . and a pretreatment with hexane ( 100 %) were analyzed . the effect of enzymatic pretreatment of cells with lysozyme was also tested . for lysozyme pretreatment , the cells were resuspended in buffer ( 20 % sucrose , 10 mm tris ph 8 . 1 , 10 mm edta , 50 mm nacl ) with lysozyme ( 2 mg / ml ) and incubated at 55 ° c . for 15 minutes . after the chemical and enzymatic pretreatments , the washing steps were the same as the washing steps used for the tca treated cells . tca pretreatment of bacillus subtilis , lactobacillus casei and mycobacterium smegmatis was done as described herein for l . lactis . cell - free culture supernatants containing msa2 :: ca , msa2 :: cd or msa2 without anchor were incubated in four - fold excess for 10 minutes at room temperature with pretreated cells ( e . g ., cells from 0 . 5 ml culture were incubated with 2 . 0 ml culture supernatant ). after binding , cells were sedimented by centrifugation , washed twice in 2 volumes demineralized water , resuspended in sds - denaturation buffer , heated for 5 minutes at 98 ° c ., subjected to sds - page , and analyzed by western blot analysis . storage conditions . cell - free supernatants containing msa2 :: ca , msa2 :: cd or msa2 were stored at − 20 ° c . with or without 10 % glycerol prior to binding . tca pretreated l . lactis cells were stored at − 80 ° c . in 10 % glycerol prior to binding . tca pretreated l . lactis cells with bound msa2 :: ca were stored at + 4 ° c . or − 80 ° c . with or without 10 % glycerol . cells stored in 10 % glycerol were washed once with 1 volume of demineralized water prior to binding . cell pellets ( in demineralized water ) of tca pretreated l . lactis cells with or without bound msa2 :: ca were frozen by contacting the vials with liquid nitrogen and removing the water with lyophilization . alternatively , non - frozen cell pellets were dried under vacuum at 30 ° c . for 2 hours prior to binding . western blotting . for detection of msa2 proteins , cell pellets corresponding to 500 μl culture were resuspended in 50 μl sds - denaturation buffer . cell - free culture supernatants ( 1 ml ) were concentrated by phenol - ether precipitation ( sauve et al . 1995 ), vacuum dried and resuspended in 50 μl sds - denaturation buffer . proteins were separated with standard sds - page techniques . after separation , proteins were electroblotted onto pvdf membranes ( roche ). in immunoblots , msa2 proteins were detected with 1 : 10 , 000 diluted rabbit msa2 - specific antiserum ( ramasamy et al . 1999 ) and 1 : 5 , 000 diluted anti - rabbit igg - conjugated alkaline phosphatase ( roche ) using known procedures . fluorescence microscopy . 100 μl cell suspensions incubated with msa2 :: ca , msa2 :: cd or msa2 fusion proteins were washed twice with demineralized water and resuspended in an equal volume pbs containing 1 % bsa and msa2 - specific rabbit antiserum diluted to 1 : 200 . after incubation for 20 minutes at room temperature , the cells were washed three times with 2 volumes pbs . subsequently , the cells were incubated for 20 minutes in 1 volume pbs with 1 % bsa and 1 : 100 diluted oregon green labeled goat anti - rabbit immunoglobulin g ( molecular probes ). after washing once with 2 volumes pbs and twice with 2 volumes demineralized water , the cells were resuspended in 100 μl demineralized water . a 10 μl aliquot of the resuspended cells was spread onto a polysin microslide ( menzel - glaser ), air dried , and examined under a fluorescence microscope ( zeiss ). electron microscopy . tca - pretreated l . lactis cells incubated with msa2 :: ca , msa2 :: cd or msa2 were collected and washed as described herein . immunogold labeling was performed on whole mount preparations of glutaraldehyde fixed cells on formvar - carbon coated nickel grids using auroprobe 15 nm goat anti - rabbit igg gold marker ( amersham ). primary antibodies against msa2 were diluted 1 : 1000 in pbs - glycine buffer . the labeled samples were stained with 0 . 1 % uranyl acetate ( w / v in water ) and examined in a philips cm10 transmission electron microscope at 100 kv . pretreatment of l . lactis cells with different chemicals . the ca protein anchor of l . lactis acma can be used to bind fusion proteins to a wide variety of gram - positive bacteria . however , the amount of fusion protein that binds varies greatly among this group of bacteria . binding of msa2 :: ca that covers the entire cell surface of some lactobacilli was observed , whereas other bacteria such as l . lactis showed only limited localized binding ( fig2 a ). this phenomenon may be due to the fact that the cell walls of some bacterial species contain components that interfere with ca anchor binding . since chemicals like sds , tca , chloroform / methanol and others may be used to remove components from isolated bacterial cell walls ( morata de ambrosini et al . 1998 ), the effect of the removal of cell - wall components from l . lactis whole cells on the binding of the reporter fusion protein msa2 :: ca was investigated . l . lactis cells were pretreated as described herein with various chemicals or with lysozyme . [ 0076 ] fig3 shows typical western blots of pretreated whole cells to which msa2 :: ca was bound . mature msa2 :: ca migrates at a position of a 75 kda protein ( indicated by an asterisk ). the arrow represents msa2 :: ca that contains the prtp prosequence . the double asterisks represent msa2 :: ca from which one or two of the repeats have been removed . a cell membrane anchored protease htra has been shown to be involved in processing proproteins and in removing repeats from acma ( poquet et al . 2000 ). from the results of fig3 it may be concluded that pretreatment with tca ( lanes 8 and 16 contain the same samples , the difference in signal intensity is due to differences in stain developing time ), hcl , h 2 so 4 and hac substantially improves the subsequent binding of msa2 :: ca ( compare with the negative control in lane 15 ). other tested acids , tfa and mca , had similar effects ( not shown ). phenol , gnhcl , formamide and chloroform -/ methanol pretreatments showed a moderate improvement of binding ( lanes 4 , 5 , 6 , 7 , respectively ). minor binding improvements were observed after pretreatment with sds , dmf , dmso and dtt . the results are summarized in table 1 . based on the results , it appears that pretreatment of l . lactis cells with the acids tca , tfa , mca , hcl , h 2 so 4 and hac are the most effective agents for improving binding of ca anchor fusion proteins to lactococcal cells . acids such as tca are known to remove lipoteichoic acids from cell walls . whether proteins are removed from the cell walls by these acid treatments was also analyzed . fig4 shows a coomassie stained gel of lysed pretreated cells . most of the acid treatments , except for hac , removed a substantial amount of proteins from the lactococcal cells . since hac removed only trace amount of proteins ( compare lane 1 and 4 ) and sds pretreatment ( which is known to remove proteins from the cell walls ) showed only a minor improvement of msa2 :: ca binding ( fig3 lane 1 ), it may be concluded that removal of proteins from the cell wall is not critical for improving the binding of ca anchor fusions . this conclusion may be due to the fact that lipoteichoic acids or carbohydrates occupy sites in the cell walls of l . lactis that interfere with efficient binding . alternatively , acid pretreatment may result in altering the compactness of peptidoglycan strands that make ca binding sites more available . tca pretreatment was also used in all other experiments . the optimal tca concentration in the boiling procedure was determined . tca percentages of 1 , 5 , 10 and 20 % were tested . although 1 % tca pretreatment already showed a significant improvement in binding of msa2 :: ca and 5 % tca pretreatment showed a further increase , no further improvement was observed at concentrations higher than 10 % tca ( fig5 ). therefore , the boiling procedure with 10 % tca was selected as the standard procedure for the experiments . the binding characteristics of the lactococcal ca homolog cd in a msa2 fusion were analyzed using the standard tca pretreatment procedure . two of the three acmd repeats are highly homologous to those of acma . an alignment is shown in fig6 . secreted msa2 without an anchoring domain was included in these experiments as a negative control . in western blots , the effect of tca pretreatment on the binding of msa2 :: ca was evident ( fig7 compare lanes 1 and 4 ). the effect of tca pretreatment was also studied using fluorescence microscopy ( fig2 compare l . lactis in a and b ; fig8 ) and electron microscopy ( fig9 compare a and b ). independent of the technique used , the effect of tca pretreatment on the binding of msa2 :: ca can be detected . the binding of msa2 :: cd to non - tca pretreated l . lactis cells was low as detected in western blots ( fig7 lane 2 ) and was undetectable in fluorescence microscopy and electron microscopy ( fig9 a ). tca pretreatment only had minor effects on the intensity of the msa2 :: cd signal in western blots ( fig7 lane 5 ). at the same time , no msa2 :: cd specific signal associated with the pretreated cells could be observed in fluorescence microscopy ( fig8 ) and only low levels of labeling was observed in electron microscopy ( fig9 c ). some cell - associated signal was observed for msa2 without anchoring domain for both non - tca pretreated and tca pretreated l . lactis cells ( fig7 lanes 3 and 6 , respectively ). however , for msa2 :: cd , this was not observed in fluorescence microscopy ( not shown ) and only minor labeling signals were found in electron microscopy ( fig9 d ). taken together , it may be concluded that : ( i ) the reporter protein msa2 does have some low degree of affinity for bacterial cell walls that can be detected in western blots ; ( ii ) the ca anchor domain specifically stimulates the binding of the reporter fusion to non - pretreated cells ; ( iii ) chemical pretreatment , especially with acids , enhances this binding ; and ( iv ) the cd anchor domain does not promote binding of fusion proteins under the conditions applied . the fluorescence microscopic images and electron microscopic images of tca pretreated lactococcal cells ( fig2 and 9 ) showed that pretreatment leaves the integrity of the cell intact . however , cells are no longer viable ( plating efficiency 0 ) and therefore may be considered as inert spherical peptidoglycan microparticles with a diameter of approximately 1 μm , “ ghost cells ”. binding to other gram - positives . the binding of msa2 :: ca , msa2 :: cd and msa2 without anchor domain to the gram - positive bacteria b . subtilis , lb . casei and m . smegmatis were also analyzed . fig1 shows a western blot summarizing binding of msa2 :: ca , msa2 :: cd and msa2 to non - pretreated and tca - pretreated b . subtilis cells . as for l . lactis , an increase in binding is observed for msa2 :: ca . a msa :: ca specific signal could also be visualized in fluorescence microscopy of non - pretreated b . subtilis cells , but with a highly improved signal for the tca - pretreated cells ( not shown ). binding of msa2 :: cd and msa2 to non - pretreated or tca - pretreated cells could not be demonstrated in fluorescence microscopy ( not shown ). similar results were obtained for lb . casei and m . smegmatis . the improved binding of msa2 :: ca to tca - pretreated lb . casei cells is shown in fig1 . for msa2 :: cd and msa2 , no fluorescence signals were detected ( not shown ). the tca - pretreatment of m . smegmatis also had a positive effect on the binding of msa2 :: ca , whereas no binding was observed for msa2 :: cd or msa2 ( fig1 ). taken together , it may be concluded that acid pretreatment , such as with tca , improves the binding of ca protein anchor fusions to the cell surface of gram - positive bacteria . binding strength and storage conditions . the strength of the msa2 :: ca binding to tca - pretreated l . lactis cells was analyzed with a treatment of licl after the binding . licl is commonly used to remove proteins from bacterial cell walls . from the western blot of fig1 , it may be concluded that 8 m licl partially removes msa2 :: ca from the l . lactis cells ( compare lanes 4 and 5 ). therefore , although msa2 :: ca binds non - covalently to cell walls , the binding interactions are most likely very strong . cell - free culture supernatants with msa2 :: ca were stored with or without 10 % glycerol at − 20 ° c . msa2 :: ca stored in this manner for several weeks had the same capacity to bind to tca - pretreated l . lactis cells ( not shown ). tca - pretreated l . lactis cells with bound msa2 :: ca were stored for 3 weeks at + 4 ° c . in demineralized water or at − 80 ° c . in demineralized water with or without 10 % glycerol . the samples were analyzed in western blots . storing pretreated cells with bound msa2 :: ca for 3 weeks in water at + 4 ° c . only resulted in a loss of signal of about 50 % ( fig1 , compare lanes 4 and 6 ). whether this loss of signal was due to degradation or due to release of the protein into the water was not determined . storage at − 80 ° c . with or without 10 % glycerol had no effect on the binding ( fig1 , compare lanes 4 , 7 and 8 ). in addition , the effects of drying and lyophilization on the binding of msa2 :: ca to tca - pretreated l . lactis cells were studied . drying of pretreated cells had no observable negative effect on binding of msa2 :: ca afterwards . dried pretreated cells with bound msa2 :: ca could be resuspended in water without losing bound fusion protein . this was also observed for lyophilized cells with bound msa2 :: ca . lyophilization of tca - pretreated cells prior to binding resulted in loss of the binding capacity for msa2 :: ca ( results not shown ). from these data , it may be concluded that : ( i ) in spite of the non - covalent character of ca anchor binding to cell walls , the binding is very strong ; ( ii ) cell - free culture supernatants can be stored safely at − 20 ° c . ; and ( iii ) drying of tca - pretreated cells provides an efficient and simple method for storage of such cells either with or without bound ca - anchor fusions . oral immunizations of rabbits with non - recombinant lactococcus lactis preloaded with the plasmodium falciparum malaria antigen msa2 fused to the lactococcal acma protein anchor . in example 1 , a technology is described that efficiently binds protein hybrids when externally added to the cell surface of non - recombinant gram - positive bacteria by means of an acma - type protein anchor . this technology provides the possibility to provide bacteria or bacterial cell walls with new traits without introducing recombinant dna into them . the immunogenicity in rabbits of the plasmodium falciparum merozoite surface protein , msa2 of strain 3d7 ( ramasamy et al . 1999 ), presented on the cell surface of non - recombinant non - living l . lactis cells as an acma anchor fusion protein was investigated . bacterial strains and growth conditions . the l . lactis strain which produces msa2 :: ca , the strain &# 39 ; s growth conditions , the induction for expression , the tca pretreatment of the l . lactis recipient cells and the binding of msa2 :: ca to the cells was described in example 1 with the following modification : a ratio of 1 ( tca - pretreated cells ) to 5 ( cell - free culture supernatant with msa2 :: ca ) was used for binding . an l . lactis nz9000 strain carrying plasmid png3043 was used as a positive control in the immunization experiments ( was positive in a previous unpublished experiment ). plasmid png3043 encodes an msa2 hybrid protein that contains the lactococcal prtp cell - wall anchoring domain at its c - terminus ( msa2 :: cp ) instead of the acma protein anchor . the prtp cell - wall anchoring domain contains the lpxtg ( seq id no : 1 ) motif that enables a membrane - linked sortase to covalently couple the protein to the cell wall ( navarre and schneewind 1994 ). the cp domain used in construct png3043 corresponds to nt 6539 to 6914 in kok et al . ( 1988 ). primers used for the amplification of this fragment were prtp . cwa . fw3 ( 5 ′- atataaagcttgcaaagtctgaaaacgaagg ( seq id no : 8 )) and prtp . cwa . rev ( 5 ′- ccgtctcaagctcactattcttcacgttgtttccg ( seq id no : 9 )). the primers include restriction endonuclease recognition sites for cloning . plasmid png3043 differs from plasmid png3041 in the cell - wall binding domain . growth conditions and induction of expression of strain nz9000δacma ( png3043 ) were the same as for strain nz9000 δacma ( png3041 ). rabbit immunizations . ten barrier - reared , new zealand white rabbits obtained from harlan laboratories , the netherlands , were used in groups of 2 for experimental immunizations . the care and use of animals were according to who guidelines ( who / lab / 88 . 1 ). the rabbits were ear bled prior to immunization to obtain preimmune sera . details of the rabbits and immunogens are as follows : rabbits a1 and a2 were subcutaneously immunized with nz9000δacma ( png3041 ) cells ( recombinant , msa2 :: ca partly surface anchored ). rabbits b1 and b2 were subcutaneously immunized with nz9000δacma ( negative control ). rabbits c1 and c2 were orally immunized with nz9000δacma ( png3043 ) cells ( recombinant , msa2 :: cp surface anchored ). rabbits d1 and d2 were orally immunized with nz9000δacma ( png3041 ) cells ( recombinant , msa2 :: ca surface anchored ). rabbits e1 and e2 were orally immunized with tca treated nz9000δacma to which msa2 :: ca had been bound from nz9000δacma ( png3041 ) culture supernatant ( non - recombinant , msa2 :: ca surface anchored ). stocks of nz9000δacma ( png3043 ) with msa2 :: cp expressed at its surface were stored in aliquots of 10 11 cells in growth medium containing 10 % glycerol at − 80 ° c . the cells remain viable under these conditions and retain msa2 on the surface as demonstrated by immunofluorescence ( not shown ). the first immunization was carried out with freshly grown bacteria . for subsequent immunizations , stocks of bacteria were freshly thawed , washed and resuspended in buffer at the appropriate concentration for immunizations . on the other hand , the non - pretreated nz9000δacma ( negative control ), the non - pretreated nz9000δacma ( png3041 ) and the tca - pretreated nz9000δacma with the externally bound msa2 :: ca were prepared daily from fresh cultures . subcutaneous injections were performed with a total of 5 × 10 9 cells in 100 μl pbs without any adjuvant into two sides on either side of the spine . the subcutaneous injections were repeated two more times at 3 week intervals . prior to oral immunization , the rabbits were deprived of water and food for 2 - 4 hours . the rabbits were then fed 5 × 10 10 cells resuspended in 1 ml of 0 . 5 % sucrose . each dose was repeated for three successive days to obtain reproducible oral immunization . altogether , three series of oral immunizations were given at 3 week intervals . adverse effects consequent to the immunizations , including granulomas at the sites of subcutaneous injections , were not observed indicating that l . lactis was well tolerated by the animals . serum antibody responses . rabbits were ear bled 2 weeks after each immunization to obtain sera for antibody assays . the sera were stored at − 20 ° c . until use . ten - fold serial dilutions of the antisera in 2 % bsa in pbs were used in immunofluorescence assays ( ifa ) to determine the titer of the antibodies against msa2 on the surface of 3d7 p . falciparum merozoites . ifa was performed on acetone - methanol fixed late stage 3d7 p . falciparum parasites as previously described ( ramasamy 1987 ). for detection of antibody isotypes , oregon green conjugated goat anti - rabbit ig ( molecular probes ) was used as the second antibody . for detection of igg antibodies , a fluorescein conjugated , affinity purified , mouse monoclonal with specificity against rabbit igg chains ( rockland ) was used . surface expression of msa2 in different l . lactis strains . coomassie staining of sds - page gels and fluorescence microscopy were used to determine , in a semi - quantitative way , the number of msa2 molecules expressed and surface exposed by the recombinant lactococcal strains carrying plasmid png3041 or png3043 that produce msa2 :: ca or msa2 :: cp , respectively , and by the non - recombinant tca - pretreated l . lactis cells to which msa2 :: ca had been bound from the outside . the recombinant strains were estimated to produce approximately 1 . 4 × 10 5 molecules of msa2 :: ca or msa2 :: cp . the surface exposure of msa2 :: ca and msa2 :: cp differed considerably as shown by fluorescence microscopy in fig1 . the non - recombinant tca - pretreated l . lactis cells with bound msa2 :: ca showed a uniform staining of the entire cell surface . however , the semi - quantitative sds - page analysis indicated that about 1 × 10 4 molecules of msa2 :: ca per cell were represented . accordingly , it may be concluded that the number of surface exposed msa2 :: ca and msa2 :: cp on the recombinant lactococcal strains is less than 10 % of the total number of molecules produced by these strains . the other molecules are most likely trapped in the membrane or the cell wall . similar observations were made by norton et al . ( 1996 ) for the expression of ttfc fused to the cp cell - wall anchoring domain . in that study , only membrane - associated or cell - wall - associated ttfc could be demonstrated and no surface - exposed ttfc :: cp was demonstrated . thus , it appears that binding from the outside to tca - pretreated cells is a more efficient method to surface - expose proteins on l . lactis cells . anti - msa2 antibody responses in orally immunized rabbits . characteristics of the anti - msa2 antibody response to the immunizations are summarized in table 2 . the oral immunizations with the recombinant l . lactis that produces msa2 :: cp ( rabbits c1 and c2 ) were done before ( unpublished results ) and used as a positive control . in the previous experiment , a similar antibody response was found . the present experiment showed that specific antibodies against near native msa2 were detectable after two immunizations for group a , d and e rabbits , and that antibody titers increased in all instances after a third immunization . igg antibodies were predominant after three immunizations in either the subcutaneous or oral route . a comparatively weak anti - msa2 surface ifa , attributable to the generation of cross - reactive antibodies ( as described herein ), was also observed after three control subcutaneous immunizations with l . lactis cells alone . taken together , the results indicate that : ( i ) msa2 produced by lactococcal cells elicits serum antibodies that recognize native p . falciparum parasite msa2 ; ( ii ) msa2 - specific t h cells are activated through mucosal immunization due to the presence of systemic igg antibodies ( table 2 ) that can be boosted ( unpublished results ); and ( iii ) oral immunizations with msa2 :: ca bound to non - recombinant non - living tca - pretreated l . lactis cells are as efficient in evoking specific serum antibody responses as the live recombinant strain producing msa2 :: ca that was administered subcutaneously or orally , or as efficient as the live recombinant strain producing msa2 :: cp that binds msa2 covalently to its cell wall delivered orally . anti - lactococcal antibody responses . western blots ( fig1 ) demonstrated significant antibody responses against l . lactis antigens after two and three immunizations of the rabbits . the responses were notably greater after subcutaneous ( group a and b rabbits ) than oral immunization with l . lactis ( group . c rabbits ). oral immunization with the tca - pretreated lactococcal cells ( group e rabbits ) elicited antibodies that reacted at a lower intensity with fewer l . lactis antigens than oral immunization with viable l . lactis cells . this is most likely due to the fact that proteins are removed from the lactococcal cells by the tca pretreatment ( see , example 1 ). the lower anti - carrier response observed for the tca - pretreated ( non - recombinant ) cells renders this type of delivery vehicle more suitable for repeated immunization strategies than its untreated ( recombinant ) counterpart . the cell - wall binding domain or anchor of the lactococcal cell - wall hydrolase acma includes three repeats of 45 amino acids that show a high degree of homology ( buist et al . 1995 ). these three repeats belong to a family of domains that meet the consensus criteria as defined in pct publication wo 99 / 25836 and can be found in various surface located proteins in a wide variety of organisms . another feature that most of these domains have in common is that their calculated pi values are high , approximately 8 or higher ( table 3 ). the ph used in previous binding experiments with msa2 :: ca ( i . e ., examples 1 and 2 ) was approximately 6 , indicating that the binding domain was positively charged . the acma protein anchor homolog of the lactococcal cell - wall hydrolase acmd ( cd ) ( bolotin et al . 2001 ) also includes three repeats ( fig1 ) with a calculated pi that is lower ( approximately pi 3 . 8 ) than that of the ca domain ( table 4 ). consequently , the cd anchor was negatively charged at the binding conditions used in example 1 . no binding of the msa2 :: cd reporter protein occurred under these conditions as demonstrated herein . therefore , the influence of the ph during binding of a cd fusion protein ( msa2 :: cd ) was investigated . furthermore , a hybrid protein anchor including the three cd repeats and one ca repeat that has a calculated pi value that is higher than that of the cd repeats alone was constructed . the hybrid protein anchor showed better binding ph values above the pi of the cd repeats alone , indicating that the ph binding range of acma - type protein anchors can be manipulated by using the pi values of the individual repeats in hybrids . bacterial strains , growth and induction conditions , tca pretreatment of l . lactis cells , incubation of the msa2 protein anchor fusion proteins to tca - pretreated cells , washing conditions , protein gel electrophoresis , western blotting and immunodetection were the same as described herein with reference to example 1 . the cell - free culture supernatants with msa2 :: ca , msa2 :: cd or a3d1d2d3 have a ph of approximately 6 . 2 . the influence of ph was examined by adjusting the ph of the cultures by the addition of hcl or naoh to obtain the required ph . plasmid constructions . the plasmid that expresses the msa2 :: cd fusion was described herein with reference to example 1 . plasmid ppa43 is based on the same expression plasmid and contains an in frame fusion of the lactococcal signal sequence of usp45 ( ssusp ; van asseldonk et al . 1990 . gene 95 : 155 - 160 ), the c - myc epitope for detection purposes , the a3 ca repeat and repeats d1 , d2 and d3 of cd . primers used for cloning a3 were carepeat3 . fw ( ccg tct cca att caa tct gct gct gct tca aat cc ( seq id no : 10 )) and ca repeat3 . rev ( taa taa gct taa agg tct cca att cct ttt att cgt aga tac tga cca att aaa ata g ( seq id no : 11 )) ( the primers include the a3 specific sequences ). the primers used for cloning the three cd repeats were cdrepeat1 . fw ( ccgtctccaatttcaggaggaactgctgttacaactag ) ( seq id no : 12 ) and cdrepeat3 . rev ( taataagcttaaaggtctccaattccagcaacttgcaaaacttctcct ac ) ( seq id no : 13 ) ( the primers include the cd specific sequences ). binding of msa2 :: cd at low ph . since binding of msa2 :: cd was not observed at a ph ( the ph of the culture medium after - growth and induction is about 6 . 2 ) higher than the calculated pi for the cd domain ( i . e ., pi 3 . 85 ), binding was studied when the ph of the medium was adjusted to ph 3 . 2 . tca - pretreated l . lactis cells were used as the binding substrate and the relative amounts of bound msa2 :: cd were analyzed in western blots . the amounts of unbound reporter protein remaining in the culture supernatant after binding were also analyzed . fig1 shows a clear increase in bound msa2 :: cd when binding is performed at ph 3 . 2 ( compare lanes 1 and 3 ). at the same time , less unbound reporter protein remained in the supernatant ( compare lanes 2 and 4 ). this result indicates that positive charges are important for binding of ca - type anchoring domains . binding of cacd hybrid anchors . analysis of the pi values of the ca homologs in table 3 indicates that two classes of repeats can be distinguished : a majority ( 99 out of 148 ) of homologs that have a high pi value (& gt ; 8 ) and a smaller group ( 33 out 148 ), of which cd is a representative , that has pi values lower than 6 . based on the experimental results , it is shown that these types of anchoring domains only bind to bacterial cell walls at a ph that is lower than the anchoring domains pi . notably , most cell - wall binding domain homologs include repeats with a pi that are representatives of one of the two groups , i . e ., only repeats with a high or low pi . some proteins with cell - wall binding domains , e . g ., those of dnir of trepanoma pallidum and an amidase of borrelia burgdorferi , include repeats with high and low pi . since the binding ph of such “ natural hybrid ” cell - wall binding domains is below the intermediate pi value of the total number of repeats present in the domain , a hybrid cell - wall protein anchor was constructed using the ca and cd repeats with an intermediate pi value . table 5 lists the native acma and acmd anchors and a number of examples of ca / cd hybrids . the constructed hybrid protein anchor ( a3 d1d2d3 ) has a calculated pi value of approximately 5 . 1 . a protein anchor including only d1d2d3 shows little binding at a ph above its calculated pi ( as described herein ). the a3 ( pi 10 ) domain shows similar binding at ph 5 and ph 7 . the binding of the hybrid anchor a3d1d2d3 was tested at ph 3 , ph 5 and ph 7 . at ph 3 , most protein had been bound to the ghost cells ( fig1 ). at ph 5 , there was considerable binding (± 40 %), whereas there was only minimal binding at ph 7 (± 20 %). this result indicates the ph range of binding for ed repeats was shifted to higher ph values by the addition of one ca repeat ( a3 ) that caused a shift in calculated pi values of 3 . 8 to 5 . 1 . the increase of binding at ph 5 for the a3d1d2d3 hybrid cannot be attributed to binding of the a3 repeat alone . if this was the case , then the same level of binding should occur at ph 7 since the a repeats show the same binding at these ph values . in addition , the increased binding at ph 5 is not an additive effect in the sense that an extra binding domain results in increased binding . it has previously been shown that addition of one repeat to the ca anchor did not result in increased binding . the binding at the higher ph values of the a3d1d2d3 repeats as compared to the d1d2d3 repeats alone , thus may be attributed to the increase in the calculated pi value of the hybrid ca / cd anchor . this demonstrates that ph binding properties of these types of protein anchors may be manipulated on the basis of the pi values of individual repeats present in the hybrid anchor . induction of cellular immune responses in mice after oral immunizations with lactococcal ghosts displaying the malaria plasmodium falciparum antigen msa2 fused to the lactococcal acma protein anchor . non - genetically modified non - living lactococcus lactis cells ( ghosts ) preloaded with the plasmodium falciparum msa2 antigen fused to the acma protein anchor ( msa2 :: ca ) were used to orally immunize mice in a similar way as described herein with reference to example 2 . in this experiment , the question of whether immunizations through the oral route with the non - recombinant non - living ghosts carrying msa2 :: ca on their surface ( ghosts - msa2 :: ca ) can elicit typical th1 - type immune responses , such as igg2 antibodies and gamma - interferon ( γifn ) producing t cells in the spleen is addressed . these responses are particularly relevant to obtain immunity for pathogens , such as malaria , that undergo stages in their life cycle where they are not in the blood , but hide in cells . groups of five mice of different strains were used for immunization . the strains of mice used were balb / c ( with the major histocompatibility locus allotype of h2d ), c57 black ( h2b ), c3h ( h2k ) and icr ( out bred , i . e ., of varying h2 types ). oral immunizations were performed at three weekly intervals . immunizations were performed with msa2 :: ca absorbed on to the surfaces of tca treated lactococcus lactis cells ( ghosts - msa2 :: ca ) or with recombinant l . iactis that displayed msa2 on the surface through the use of a covalently linked cell - wall anchor ( l . lactis ( msa2 :: cp )) as described herein with reference to example 2 . the mice were tail bled to obtain serum samples two weeks after the second , third and fourth immunizations . fecal pellets were collected and extracted to examine intestinal iga antibody production . the mice were sacrificed at the end of each experiment and the spleens were removed for examining t - cell responses by elispot . msa2 - his tag produced in e . coli was used as antigen in the elisa and elispot assay . the growth of bacterial strains and the preparation of ghost cells was as described herein with reference to example 2 . kinetics and isotypes of the serum igg antibodies generated oral immunizations . differences in the kinetics of the antibody response and the isotype distribution were observed between different murine strains . the antibody response was also different when living recombinant l . lactis ( msa2 :: cp ) or ghosts - msa2 :: ca were used as immunogens . with ghosts - msa2 :: ca , high serum antibody levels were detectable in the c3h mice after two immunizations . igg antibodies were detectable in all four murine strains after three and four immunizations . antibody titers were highest in c3h mice . igg antibodies that reacted with native msa2 on parasites were detected in the sera of immune mice by fluorescence microscopy ( ifa ) confirming that the immunizing form of the protein elicits biologically relevant antibodies . control immunizations were performed with ghosts alone where no msa2 - specific antibodies were elicited . in parallel experiments using msa2cp as the immunogen , high serum igg antibody levels were only seen with balb / c mice after two immunizations . after three and four immunizations , good antibody responses developed in c3h mice . antibody titers were highest in balb / c mice . significant differences existed between the strains in the isotypes of the elicited serum igg antibodies in response to immunization with ghosts - msa2 :: ca . balb / c mice showed higher levels of igg2a and igg2b antibodies , some igg3 antibodies and negligible igg1 which demonstrates a possible th1 bias . on the other hand , c57 black and c3h mice had high igg1 , igg2a and igg2b , and lower igg3 antibodies to msa2 which is more characteristic of a mixed th1 and th2 response . icr mice , as expected , showed a range of responses . some icr mice had the balb / c and others the c3h / c57 black pattern of igg isotypes . formation of mucosal antibodies . iga antibodies were detected by elisa in the fecal pellets of the icr and balb / c mice , but were not detected in c3h or c57 black mice when immunization was performed with living recombinant l . lactis ( msa2 :: cp ) or ghost - msa2 :: ca . t - cell responses . the increase of the intensity of the igg elisa reactions seen in mice immunized with ghosts - msa2 :: ca with each immunization demonstrates that boosting takes place and that a th - dependent antibody response exists in these animals . the igg isotype distribution further confirms this conclusion . therefore , th cells are generated in icr , balb / c , c57 black and c3h mice . the elispot assay for detecting gamma - interferon ( γifn ) producing cells detects mainly cd8 + tc cells , which are an important component of the immune response to many pathogens , including malaria parasites . his - tagged msa2 produced in e . coli was used as antigen in the assay . msa2 - specific γifn producing cells could be detected in the spleens of balb / c , c57 black and c3h mice that were immunized with ghosts - msa2 :: ca . msa2 - specific γifn producing cells were not observed in the spleens of control mice immunized with ghosts alone or with the living recombinant l . lactis ( msa2 - cp ). the latter group showed a high level of non - specific γifn producing cells . the high background observed may be due to ongoing inflammation . the sensitization of msa2 - specific tc cells in the spleen after immunization with the non - recombinant non - living l . lactis ghost - system carrying a foreign protein is a novel finding which is applicable to malaria since protection against sporozoite - infection is associated with γifn producing cells being produced in the spleen . the non - recombinant non - living ghost system can be used in oral immunizations to elicit typical th1 - type immune responses . these types of responses are particularly relevant to obtain immunity for pathogens that undergo stages in their life cycle where the pathogens are not in the blood , but rather hide in cells . the responses are more pronounced and more specific for the ghost system than for the living recombinant system . the ghost system has the additional advantage of eliminating the risk of spreading recombinant dna into the environment . protection of mice for lethal streptococcus pneumoniae challenge after oral immunizations with lactococcal ghosts preloaded with ppma antigen fused to the lactococcal acma protein anchor . [ 0132 ] streptococcus pneumoniae is the leading etiological agent of severe infections including septicemia , meningitis , pneumonia , and otitis media . recent studies on the molecular epidemiology and pathogenesis of s . pneumoniae have identified pneumococcal proteins with vaccine potential . one of these proteins , the protease maturation protein ppma , has been shown to elicit immune protective potential in a mouse pneumonia model . the non - genetically modified lactococcal ghosts have been shown to be an efficient carrier for use in oral immunizations of rabbits and mice in order to elicit strong anti - malaria immune responses . the construction of lactococcal ghosts that display the s . pneumoniae ppma fused to the lactococcal acma cell - wall binding domain on their surface is described herein . the ability of these ghosts to protect orally immunized mice from a lethal nasal dose of s . pneumoniae was investigated . bacterial strains and growth conditions . l . lactis was grown and ghost cells were prepared as described herein with reference to example 1 . s . pneumoniae was grown as described before ( gingles et al . 2001 . infect immun 69 : 426 - 434 ). construction ppma protein anchor fusion expression plasmid . the expression plasmid for ppma protein anchor fusion ( ppma :: ca ) was substantially similar to the expression plasmid for the msa2 protein anchor fusion as described herein with reference to example 2 . for the secretion of ppma :: ca , the secretion signal sequence of the usp45 protein ( ssusp ) of l . lactis ( van asseldonk et al . 1990 . gene 95 : 155 - 160 ) was used . the ppma gene was cloned by pcr using primers ppma . 1 ( cggtctcacatgtcgaaagggtcagaaggtg cagacc ) ( seq id no : 14 ) and ppma . 2 ( cggtctcgaattgcttcgtttgatgtactactg cttgag ) ( seq id no : 15 ) resulting in plasmid ppa32 which contains ppma as an in frame fusion with ssusp45 and the protein anchor ( ssusp :: ppma :: ca ). expression of the fusion gene results in the secreted product ppma :: ca . the primers include an eco31i restriction enzyme recognition site that was used for digestion of the pcr fragment . this restriction digest produced ncoi and ecori sticky ends which were used for cloning . the primers also iuncluded the ppma sequences . chromosomal dna of s . pneumoniae strain d39 was used as a template for the pcr reactions . preparation of the vaccine . three liters of m17 medium with ppma :: ca , obtained after growth and used to induce producer cells for expression of l . lactis ( ppa32 ), was centrifuged and filter sterilized ( 0 . 2 μm ) to remove the producer cells . ghost cells were prepared from 0 . 5 liter of l . lactis nz9000 ( δacma ). after binding , the ghost cells with ppma :: ca ( ghosts - ppma :: ca ) were isolated by centrifugation and washed with pbs . the ghost cells were stored in pbs in aliquots of 2 . 5 × 10 10 ghosts / ml at ‘ 80 ° c . two control groups included : ( i ) ghosts without bound ppma :: ca ; for the sample preparation the same amounts of ghost cells were used and the same centrifugation and washing steps were performed , but the binding step was omitted ; and ( ii ) soluble ppma was isolated as a his - tagged fusion . mice immunizations . groups of 10 mice ( cd - 1 ) were used in the immunizations . oral doses included 5 × 10 9 ghosts with or without ppma :: ca ( 50 μg ) or 50 μg soluble ppma in pbs . nasal doses included 5 × 10 8 ghosts with or without ppma :: ca ( 5 μg ) or 5 μg soluble ppma . 10 8 ghosts - ppma :: ca ( 1 μg ) were subcutaneously injected . for intranasal immunizations , the mice were slightly anesthetized with isofluorane . intranasal challenge . the groups of orally immunized mice were intranasally challenged 14 days after the last booster immunization with a dose of 10 6 colony forming units ( cfu ) s . pneumoniae d39 as described ( kadioglu et al . 2000 infect immun 68 : 492 - 501 ). mice were monitored after the challenge for visible clinical symptoms for 7 days , at which point the experiment was ended . mice that were alive after 7 days were considered to have survived the pneumococcal challenge and mice that became moribund during the 7 - day period were judged to have reached the endpoint of the assay . the time the animal became moribund was recorded , and the animal was sacrificed by cervical dislocation . elisa analysis . serum samples were taken from each mouse before the intranasal challenge and stored at − 20 ° c . before use . microtiter plates were coated with 100 μg ppma / ml in 0 . 05 carbonate buffer . serial 10 - fold dilutions of pooled serum of each group were incubated on the plates as described ( gingles et al . 2001 , infect . immun . 69 : 426 - 434 ). anti - mouse immunoglobulin - horse - radish peroxidase conjugate was used for detection and the absorbance was measured at 492 nm . serum antibody response . mice were immunized orally , nasally and subcutaneously according to the scheme shown in fig1 . anti - ppma antibody titers in the blood serum were determined for each group by elisa assays . the results are given in fig2 . as expected , ghosts alone administered orally or nasally , ov ghosts or in ghosts , respectively , did not induce anti - ppma antibodies . soluble ppma given by the nasal route resulted in only a low anti - ppma antibody titer which agrees with the general findings that soluble antigens are not very immunogenic when given by the mucosal routes . ghosts - ppma :: ca provided by the oral route ( ov ppma + ghost ) induced only a low level of anti - ppma serum antibodies . this contrasts the results for the oral immunization experiments described herein with reference to examples 2 and 4 with msa2 :: ca . however , the contrast may be antigen - type related . intranasal administration of ghosts - ppma :: ca resulted in a high titer of anti - ppma antibodies ( in ppma + ghosts ). a high titer was also obtained by subcutaneous administration of ghosts - ppma :: ca . these titers were lower by a factor of 5 to 10 when compared to soluble ppma that was subcutaneously administered and formulated with the strong freunds complete adjuvant ( peter adrian , erasmus university rotterdam , the netherlands , unpublished results ). in addition , the freunds ppma vaccine contained 50 μg ppma per dose , whereas the intranasally administered ghosts - ppma : ca contains only 5 μg / dose and the subcutaneous ghost - ppma : : ca vaccine contains only 1 μg ppma / dose . this result demonstrates the adjuvant effect of the ghost cells . side effects of the orally , nasally or subcutaneously administrated ghosts were not observed , which is in contrast to the severe side effects that are usually seen with the use of freunds adjuvants . the results demonstrate that high titer serum antibodies can be obtained by the mucosal route of adminstration . these data also show that ghost cells may be safely used in traditionally injected vaccines without side effects in order to induce high titer serum antibodies . protection against challenge . the mice orally immunized with soluble ppma , ghosts alone or ghosts - ppma :: ca were challenged 14 days post immunization with a lethal intranasal dose of s . pneumoniae . the mice immunized with soluble ppma or ghosts alone died within 72 hours after challenge . the group immunized with ghosts - ppma :: ca showed a survival rate of 40 % ( fig2 ). this results shows that mucosal immunization of mice with ghosts - ppma induces protective immunity against a lethal s . pneumoniae challenge . in conclusion , the non - recombinant non - living ghost system may be used to elicit high titer serum antibodies and the mucosal route of administration may be used to obtain protective immunity against a mucosally acquired pathogen . morata de ambrosini et al . ( 1998 ) j . food prot . 61 : 557 - 562 . norton et al . ( 1996 ) fems immunol . med . microbiol . 14 : 167 - 177 . [ 0161 ] table 1 effect of different pretreatments of l . lactis on binding of msa2 :: ca . treatment signal on western blot h 2 o − 10 % tca ( 0 . 6 m ) ++++ 0 . 6 m hac ++++ 0 . 6 m hcl ++++ 0 . 6 m h 2 so 4 ++++ 0 . 6 m tfa ++++ 0 . 6 m mca ++++ phenol ++ 4 m gnhcl ++ 37 % formaldehyde ++ chcl 3 / meoh ++ 10 % sds + 10 % dmf + 10 % dmso + 25 mm dtt + 0 . 1 % nahclo * − hexane − lysozyme * − [ 0162 ] table 2 msa2 antibody titres of rabbit serum determined by ifa on plasmodium falciparum 3d7 asexual blood stage parasites . immunisation rabbit 3 rd serum immunogen p 2 nd igg a1 s . c . l . lactis [ msa2 :: ca ] 0 2 4 4 a2 s . c . l . lactis [ msa2 :: ca ] 0 2 5 5 b1 s . c . l . lactis 0 0 1 n . d . b2 s . c . l . lactis 0 0 1 n . d . c1 oral l . lactis [ msa2 :: cp ] 0 3 5 5 c2 oral l . lactis [ msa2 :: cp ] 0 2 5 5 d1 oral l . lactis [ msa2 :: ca ] 0 2 4 5 d2 oral l . lactis [ msa2 :: ca ] 0 2 4 5 e1 oral tca l . lactis + msa2 :: ca 0 2 5 5 e2 oral tca l . lactis + msa2 :: ca 0 2 5 4 [ 0163 ] table 3 acma cell wall binding domain homologs and their calculated pi values . ( the pi values are indicated directly behind the amino acid sequences ) lactococcus * acma ytvksgdtlwgisqryg seq id no : 16 9 . 75 245 - 287 ( 33 ) 437 u1769600 muramidase lactis isvaqiqsannlkst i iyigqklvlt vkvksgdtlwalsvkyk seq id no : 17 9 . 64 321 - 363 ( 31 ) tsiaqlkswnhlssd t iyigqnlivs hkvvkgdtlwglsqksg seq id no : 18 10 . 06 395 - 437 spiasikawnhlssd t iligqylrik * acmd ykvqegdslsaiaaqyg seq id no : 19 4 . 15 194 - 237 qgci25 ttvdalvsanslenand ihvgevlqva ytvksgdslysiaeqyg seq id no : 20 3 . 78 258 - 303 mtvsslmsangiydvns mlqvgqvlqvtv ytiqngdsiysiatang seq id no : 21 4 . 15 319 - 361 mtadlaalngfgind m ihpgqtiri  tuc2009 * lys yvvkqgdtlsgiasnwg seq id no : 22 6 . 31 332 - 375 ( 10 ) 428 l31364 glycosidase tnwqelarqnslsnpnm ( muramidase ) iyagqvisft ytvqsgdnlssiaillg seq id no : 23 3 . 45 386 - 428 ttvqslvsmngisnpnl iyagqtlny - lc3 * lysb yivkqgdtlsgiasnlg seq id no : 24 6 . 31 333 - 376 ( 10 ) 429 u04309 muramidase tnwqelarqnslsnpnm iysgqvislt ytvqsgdnlssiarrlg seq id no : 25 8 . 79 387 - 429 gisnpnliyagqtlny enterococcus * autolysin ytvksgdtlnkiaaqyg seq id no : 26 9 . 74 363 - 405 ( 25 ) 671 p37710 muramidase faecalis vsvanlrswngisgd l ifvgqklivk ytvksgdtlnkiaaqyg seq id no : 27 9 . 74 431 - 473 ( 25 ) vtvanlrswngisgd l ifvgqklivk ytiksgdtlnkiaaqyg seq id no : 28 9 . 74 499 - 541 ( 25 ) vsvanlrswngisgd l ifagqkiivk ytiksgdtlnkisaqfg seq id no : 29 9 . 85 567 - 609 ( 19 ) vsvanlrswngikgd l ifagqtiivk htvksgdslwglsmqyg seq id no : 30 9 . 35 629 - 671 isiqkikqlnglsgd t iyigqtlkvg hirae * mur2 ytvksgdsvwgishsfg seq id no : 31 9 . 35 257 - 299 ( 38 ) 666 p39046 muramidase itmaqliewnniknn f iypgqkltik ytvksgdsvwkiandhg seq id no : 32 7 . 14 338 - 380 ( 33 ) ismnqliewnniknk f vypgqqlvvs ytvkagesvwsvsnkfg seq id no : 33 9 . 91 414 - 456 ( 32 ) ismnqliqwnniknn f iypgqklivk ytvkagesvwgvangis seq id no : 34 9 . 64 489 - 531 ( 33 ) mnqliewnniknn fiy pgqklivk ytvkagesvwgvankhh seq id no : 35 7 . 31 565 - 607 ( 15 ) itmdqliewnniknn f iypgqevivk ytvkagesvwgvadshg seq id no : 36 7 . 15 623 - 665 itmnqliewnniknn f iypgqqlivk listeria * p60 vvveagdtlwgiaqskg seq id no : 37 8 . 61 30 - 72 ( 130 ) 484 p21171 adherence monocytogenes ttvdaikkannlttd k and invasion ivpgqklqvn protein p60 havksgdtiwalsvkyg seq id no : 38 9 . 35 203 - 245 vsvqdimswnnlsss s iyvgqklaik innocua * p60 vvveagdtlwgiaqskg seq id no : 39 8 . 61 30 - 72 ( 129 ) 481 q01836 adherence and ttvdaikkannlttd k invasion ivpgqklqvn protein p60 hnvksgdtiwalsvkyg seq id no : 40 8 . 35 201 - 243 vsvqdimswnnlsss s iyvgqkpaik ivanovii * p60 vvveagdtlwgiaqdkg seq id no : 41 6 . 35 30 - 72 ( 125 ) 524 q01837 adherence ttvdalkkannltsd k and invasion ivpgqklqit protein p60 ytvksgdtiwalsskyg seq id no : 42 9 . 37 198 - 240 ( 73 ) tsvqnimswnnlsss s iyvgqvlavk ytvksgdtlskiattfg seq id no : 43 9 . 89 314 - 356 ttvskikalnglnsd n lqvgqvlkvk seeligeri * p60 vvveagdtlwgiaqdng seq id no : 44 6 . 35 30 - 72 ( 127 ) 523 q01838 adherence ttvdalkkanklttd k and invasion ivpgqklqvt protein p60 htvksgdtiwalsvkyg seq id no : 45 8 . 64 200 - 242 ( 77 ) asvqdlmswnnlsss s iyvgqniavk ytvksgdtlgkiastfg seq id no : 46 9 . 62 320 - 362 ttvskikalngltsd n lqvgdvlkvk welshimeri * p60 vvveagdtlwgiaqskg seq id no : 47 8 . 61 30 - 72 ( 125 ) 524 m80348 adherence ttvdalkkannltsd k and invasion ivpgqklqvt protein p60 htvksgdtiwalsvkyg seq id no : 48 9 . 35 198 - 240 ( 75 ) asvqdlmswnnlsss s iyvgqkiavk ytvksgdslskiantfg seq id no : 49 9 . 89 316 - 358 tsvskikalnnltsd n lqvgtvlkvk grayi * p60 vvvasgdtlwgiasktg seq id no : 50 9 . 42 30 - 72 ( 104 ) 511 q01835 adherence ttvdqlkqlnkldsd r and invasion ivpgqkltik protein p60 ykvksgdtiwalsvkyg seq id no : 51 9 . 57 177 - 219 ( 79 ) vpvqkliewnnlsss s iyvgqtiavk ykvqngdslgkiaslfk seq id no : 52 8 . 59 299 - 342 vsvadltnwnnlnatit iyagqelsvk haemophilus * amib hivkkgeslgslsnkyh seq id no : 53 10 . 11 294 - 336 432 p44493 n - acetylmur - influenzae vkvsdiiklnqlkrk t amoyl - l - lwlnesikip alanine amidase hkvtknqtlyaisreyn seq id no : 54 10 . 49 387 - 430 ipvnillslnphlkng k vitgqkiklr * yeba ytvtegdtlkdvlvlsg seq id no : 55 3 . 87 131 - 174 475 p44693 homologous to lddssvqplialdpela endopeptidase of hlkagqqfywi staphylococcus lppb ykvnkgdtmfliaylag seq id no : 56 6 . 40 147 - 190 405 p44833 outer idvkelaalnnlsepny membrane nlslgqvlkis lipoprotein somnus lppb ykvrkgdtmfliayisg seq id no : 57 8 . 56 120 - 164 279 l10653 outer membrane mdikelatlnnmsepy lipoprotein hlsigqvlkia helicobacter dnir hvvlpketlssiakry seq id no : 58 10 . 02 319 - 361 372 ae000654 regulatory pylori qvsisniqlandlkds n protein dnir ifihqrliir pseudomonas lppb yivrrgdtlysiafrfg seq id no : 59 10 . 06 69 - 113 297 p45682 lipoprotein aeruginosa wdwkalaarngiappyt iqvgqaiqfg putida nlpd yivkpgdtlfsiafrygw seq id no : 60 9 . 77 44 - 87 244 y19122 lipoprotein dykelaarngipapytir pgqpirfs sinohizobium nlpd imvrqgdtvtvlarrfgv seq id no : 61 10 . 27 166 - 209 512 u81296 lipoprotein meliloti pekeilkanglksasqv epgqrlvip synechocystis nlpd hqvkegeslwqisqafq seq id no : 62 4 . 38 87 - 130 715 d90915 lipoprotein sp . vdakaialansistdte lqagqvlnip slr0878 hvvkagetidsiaaqyq seq id no : 63 5 . 41 4 - 47 245 d90907 hypothetical lvpatlisvnnqlssgq protein vtpgqtilip aqiufex nlpdl ykvkkgdslwkiakeyk seq id no : 64 10 . 08 26 - 70 ( 24 ) 349 ae000700 lipoprotein aeolicus tsigkllelnpklknrk ylrpgekiclk yrvkrgdslikiakkfg seq id no : 65 10 . 95 95 - 137 ( 37 ) vsvkeikrvnklkgn r iyvgqklkip yrvrrgdtlikiakrfr seq id no : 66 12 . 11 174 - 216 tsvkeikrinrlkgn l irvgqklkip volvox ytiqpgdtfwaiaqrrg seq id no : 67 9 . 03 42 - 85 309 af058716 chitinase carteri ttvdviqslnpgvvptr lqvgqvinvp f . nagariensis ytiqpgdtfwaiaqrrg seq id no : 68 9 . 03 106 - 149 ttvdviqslnpgvnpar lqvgqvinvp staphylococcus prota hvvkpgdtvndiakang seqidno : 69 5 . 58 431 - 474 524 a04512 protein a aureus ttadkiaadnkladmik pgqelvvd lytn ytvkkgdtlsaialkyk seq id no : 70 10 . 03 177 - 220 383 af106851 autolysin ttvsniqntnnianpnl homolog ifigqklkvp colletotrichum cihl hkvkgeslttiaekydt seq id no : 71 4 . 76 110 - 153 ( 31 ) 230 aj001441 glycoprotein gicniaklnnladpnfi dlnqdlqip lindemuthianum ysvvsgdtltsiaqalq seq id no : 72 5 . 46 185 - 228 itlqslkdanpgvvpeh lnvgqklnvp chlamydophda amib ivyregdslskiakkyk seq id no : 73 9 . 46 159 - 201 205 ae001659 n - acetylmur - lsvtelkkinkldsd a . 1 amoyl - l - ala iyagqrlclq amidase pneumoniae cpn0593 yvvqdgdslwliakrfg seq id no : 74 10 . 01 316 - 358 362 ae001643 ipmdkiiqknglnhh r lfpgkvlklp nlpd vvvkkgdfleriaranh seq id no : 75 10 . 17 124 - 166 233 ae001670 muramidase ttvaklmqindlttt q lkigqvikvp yivqegdspwtialrnhi seq id no : 76 8 . 64 188 - 233 rlddllkmndldeykar rlkpgdqlfir chlamydia nlpd vivkkgdfleriarsnh seq id no : 77 9 . 99 138 - 180 245 ae001348 muramidase trachomatis ttvsalmqlndlsst q lqigqvlrvp yvvkegdspwaialsng seq id no : 78 10 . 00 200 - 245 irldellklngldeqka rrlrpgdrlrir papq hivkqgetlskiaskyn seq id no : 79 9 . 89 155 - 197 200 ae001330 ipvvelkklnklnsd t iftdqrirlp prevotella phg htvrsneslydisqqyg seq id no : 80 10 . 72 266 - 309 309 af017417 hemagglutinin intermedia vrlknimkanrkivkrgi kagdrvvl leuconostoc lys ytvqsgdtlgaiaakyg seq id no : 81 9 . 23 335 - 378 432 endolysin oenos  10mc ttyqkiaslngigspy iiipgeklkvs ykvasgdtlsaiaskyg seq id no : 82 9 . 68 389 - 432 tsvsklvslnglknany iyvgenlkik oenococcus lys44 ytvrsgdtlgaiaakyg seq id no : 83 9 . 58 335 - 378 432 af047001 lysin oeni  fog44 ttyqklaslngigspyi iipgeklkvs ykvasgdtlsaiaskyg seq id no : 84 9 . 95 389 - 432 tsvsklvslnglknany iyvgqtlrik thermotoga tm0409 ykvqkndtlysislnfg seq id no : 85 5 . 49 26 - 69 271 ae001720 maritime ispsllldwnpgldphs lrvgqeivip ytvkkgdtldaiakrff seq id no : 86 9 . 73 76 - 118 ttatfikeanqlksy t iyagqklfip tm1686 hvvkrgetlwsianqyg seq id no : 87 8 . 76 212 - 255 395 ae001809 vrvgdivlinrledpdr ivagqvlkig treponema dnir htirsgdtlyalarryg seq id no : 88 10 . 58 607 - 650 779 ae001237 membrane - pallidum lgvdtlkahnrahsath bound lytic lkigqkliip murein trans - glycosylase d hvvqqgdtlwslakrygv seq id no : 89 4 . 81 734 - 777 svenlaeennlavdatls lgmilktp tp0155 yevregdvvgriaqryd seq id no : 90 9 . 58 87 - 130 371 ae001200 isqdaiislnklrstra lqvgqllkip tp0444 hviakgetlfslsrryg seq id no : 91 10 . 98 67 - 110 342 ae001221 vplsalaqannlanvhq lvpgqrivvp borrelia bb0262 hkikpgetlshvaaryq seq id no : 92 9 . 72 183 - 226 ( 6 ) 417 ae001137 hypothetical burgdorferi itsetlisfneikdvrn protein ikpnsvikvp yivkkndsissiasayn seq id no : 93 4 . 58 233 - 275 vpkvdildsnnldne v lflgqklfip * bb0625 ykvvkgdtlfsiaikyk seq id no : 94 10 . 02 44 - 86 ( 28 ) 697 ae001164 n - acetylmur - vkvsdlkrinklnvd n amoyl - l - ikagqiliip alanine amidase ytakegdtiesisklvg seq id no : 95 5 . 00 115 - 157 ( 7 ) lsqeeiiawndlrsk d lkvgmklvlt ymvrkgdslsklsqdfd seq id no : 96 9 . 20 165 - 207 ( 8 ) isskdilkfnflndd k lkigqqlflk hyvkrgetlgriayiyg seq id no : 97 10 . 05 216 - 258 ( 27 ) vtakdlvalngnrai n lkagsllnvl hsvavgetlysiarhyg seq id no : 98 7 . 41 286 - 328 vliedlknwnnlssn n imhdqklkif bb0761 ykvkkgdtftkiankin seq id no : 99 9 . 34 59 - 102 295 ae001176 gwqsgiatinlldsp a vsvgqeilip lacrobacillus * lys ytvvsgdswnkiaqrngl seq id no : 100 9 . 83 399 - 442 442 x90511 lysin  gle smytlasqngksiysti ypgnkliik bacillus * lyte ikvkkgdtlwdlsrkyd seq id no : 101 9 . 55 28 - 70 ( 17 ) 334 u38819 . 1 d - glutamate - subtilis ttiskiksenhlrsd i m - diaminopim - iyvgqtlsin late endo - peptidase ykvksgdslwkiskkyg seq id no : 102 10 . 16 88 - 130 ( 20 ) mtinelkklnglksd l lrvgqvlklk ykvksgdslskiaskyg seq id no : 103 10 . 03 151 - 193 ttvsklkslnglksd v iyvnqvlkvk spovid civqqedtierlceryei seq id no : 104 4 . 20 525 - 568 575 p37963 stage vi spor - tsqqlirmnslalddelk ulation pro - agqilyip tein d yaah mvkqgdtlsaiasqyrt seq id no : 105 3 . 89 1 - 43 ( 5 ) 427 p37531 hypothetical ttnditetneipnpdsl protein vvgqtivip ydvkrgdtltsiarqfn seq id no : 106 10 . 32 49 - 92 ttaaelarvnriqlntv lqigfrlyip yhdd ikvksgdslwklaqtyn seq id no : 107 9 . 56 29 - 71 ( 22 ) 488 y14079 hypothetical tsvaaltsanhlstt v protein lsigqtltip ytvksgdslwlianefm seq id no : 108 9 . 62 94 - 136 ( 39 ) tvqelkklnglssd li ragqklkvs ykvqlgdslwkiankvn seq id no : 109 9 . 72 176 - 218 ( 23 ) msiaelkvlnnlksd t iyvnqvlktk ytvksgdslwkiannyn seq id no : 110 9 . 65 242 - 284 ( 24 ) ltvqqirninnlksd v lyvgqvlklt ytvsgdslwviaqkfnv seq id no : 111 9 . 72 309 - 351 taqqireknnlktd vl gvgqklvis yojl ikvksgdslwklsrqyd seq id no : 112 9 . 93 29 - 71 ( 18 ) 414 z99114 similar to ttisalksenklkst v cell wall lyvgqslkvp binding protein ytvaygdslwmiaknhm seq id no : 113 9 . 81 90 - 132 ( 26 ) svselkslnslssd li rpgqklkik ytvklgdslwkiansln seq id no : 114 9 . 27 159 - 201 ( 25 ) mtvaelktlngltsd t lypkqvlkig ykvkagdslwkianrlg seq id no : 115 9 . 84 227 - 269 vtvqsirdknnlssd v lqigqvltis yoch itvqkgdtlwgisqkng seq id no : 116 9 . 25 28 - 70 ( 9 ) 287 af027868 similar kgvnlkdlkewnkltsd k to papq iiagekltis ytikagdtlskiaqkfg seq id no : 117 9 . 64 80 - 122 ttvnnlkvwnnlssd m iyagstlsvk ykvp hhvtpgetlsiiaskyn seq id no : 118 8 . 65 345 - 387 399 z99111 hypothetical vslqqlnelnhfksd q protein iyagqiikir * xlyb yhvkkgdtlsgiaaseg seq id no : 119 9 . 72 179 - 222 317 z99110 n - acetylmur - asvktlqsinhitdpnh amoyl - l - ikigqviklp alanine amidase yrba hivqkgdslwkiaekyg seq id no : 120 8 . 51 4 - 48 387 z99118 similar to vdveevkklntqlsnpd spore coat limpgmkikvp protein ydhd hivgpgdslfsigrryg seq id no : 121 5 . 49 4 - 46 439 z99107 asvdqirgvngldet n ivpgqallip ykud yqvkqgdtlnsiaadfr seq id no : 122 6 . 10 4 - 46 164 z99111 istaallqanpslqa g ltagqsivip  pbsx * xlya yvvkqgdtltsiapafg seq id no : 123 4 . 65 161 - 204 297 p39800 n - acetylmur - vtvaqlqewnniedpnl amoyl - l - irvgqvlivs alanine amidase  pza * orf15 ykvksgdnltkiakhn seq id no : 124 10 . 23 163 - 207 ( 6 ) 258 p11187 ttvatllklnpsikdp nmirvgqtinvt (=. 29 ) hkvksgdtlskiavdnk seq id no : 125 10 . 17 214 - 258 p07540 ttvsrlmslnpeitnpn hikvgqtirls  b103 * orfl5 hvvkkgdtlseiakki seq id no : 126 10 . 11 165 - 209 ( 9 ) 263 x99260 lysozyme ktstktllelnptikn pnkiyvgqrinvg ykikrgetltgiakkt seq id no : 127 10 . 61 219 - 263 tvsqlmklnpnikain nyagqtirlk sphaericus * pep1 ilirpgdslwyfsdlfk seq id no : 128 8 . 86 3 - 46 ( 6 ) 396 x69507 carboxypepti - iplqllldsnrninpq l lqvgqriqip dase i ytitqgdslwqiaqnkn seq id no : 129 7 . 15 53 - 96 lplnaillvnpeiqps r lhigqtiqvp salmonella nlpd ytvkkgdtlfyiawitg seq id no : 130 8 . 64 121 - 164 377 aj006131 dublin ndfrdlaqrnsisapy slnvgqtlqvg escherichia * yeba yvvstgdtlssilnqyg seq id no : 131 4 . 13 77 - 121 419 p24204 homologous to coli idmgdisqlaaadkelr endopeptidase nlkigqqlswt of staphy - lococcus mltd ytvrsgdtlssiasrlg seq id no : 132 10 . 58 343 - 385 ( 16 ) 452 p23931 membrane - vstkdlqqwnklrgs k bound lytic lkpgqsltig murein trans - glycosylase d precursor yrvrkgdslssiakrhg seq id no : 133 10 . 18 402 - 443 vnikdvmrwnsdtan l qpgdkltlf uug ytvkrgdtlyrisrttg seq id no : 134 10 . 16 50 - 93 259 u28375 hypothetical tsvkelarlngisppyt protein ievgqklklg nlpd ytvkkgdtlfyiawitg seq id no : 135 8 . 64 123 - 166 379 p33648 lipoprotein ndfrlaqrnniqapyal nvgqtlqvg drosophila q9vna1 ytvgnrdtltsvaa seq id no : 136 7 . 15 329 - 371 1325 af125384 lethal s2fd melanogaster rfdttpselthlnr protein lnss fiypgqqllvp drosophila q961p8 ytvgnrdtltsvaarfd seq id no : 136 7 . 15 104 - 146 678 aak92873 melanogaster ttpselthlnrlnss f iypgqqllvp caenorhabditis f43g9 . 2 rkvkugdtlnklaikyq seq id no : 137 10 . 01 12 - 55 179 z79755 elegans vnvaeikrvnnmvseqd fmalskvkip caenorhabditis f52e1 . 13 ytitetdtlervaashd seq id no : 138 7 . 08 24 - 66 819 u41109 elegans ctvgelmklnkmasr m vfpgqkilvp caenorhabditis f07g11 . 9 teiksgdscwniasnak seq id no : 139 8 . 32 23 - 66 ( 11 ) 1614 u64836 / putative elegans isverlqqlnkgmkcdk lplgdklcla af016419 endochitinase lklkaedtcfkiwssqk seq id no : 140 7 . 84 78 - 121 ( 21 ) lserqflgmnegmdcdk lkvgkevcva hkiqkgdtcfkiwttnk seq id no : 141 8 . 65 143 - 186 ( 21 ) isekqfrnlnkgldcdk leigkevcis lkikegdtcyniwtsqk seq id no : 142 4 . 54 208 - 251 ( 19 ) iseqefmelnkgldcdk leigkevcvt yrfkkgdtcykiwtshk seq id no : 143 9 . 35 271 - 314 ( 20 ) msekqfralnrgidcdr lvpgkelcvg itvkpgdtcfsiwtsqk seq id no : 144 4 . 21 335 - 378 ( 23 ) mtqqqfmdinpeldcdk leigkevcvt vkinpgdtcfniwtsqr seq id no : 145 6 . 30 402 - 445 ( 21 ) ntqqqfmdlnkrldcdk levgkevcva vqinpgdtcfkiwsaqk seq id no : 146 4 . 60 467 - 510 ( 37 ) lteqqfmelnkgldcdr levgkevcia tevkegdtcfkiwsahk seq id no : 147 5 . 12 548 - 591 ( 44 ) iteqqfmemnrgldcnr levgkevciv ikvkegdtcfkiwsaqk seq id no : 148 7 . 85 636 - 679 ( 66 ) mteqqfmemnrgldcnk lmvgkevcvs atitpgntcfnisvayg seq id no : 149 3 . 99 746 - 786 ( 8 ) inlt dlqktydckale vgdticvs ievikgdtcwflenafk seq id no : 150 4 . 67 795 - 838 tnqtemeranegvkcdn lpigrmmcvw caenorhabditis t01c4 . 1 htiksgdtcwkiasea seq id no : 151 5 . 01 23 - 66 ( 51 ) 1484 u70858 putative elegans sisvqeleglnskksc endochitinase anlavglseqef ihvkegdtcytiwtsqh seq id no : 152 4 . 12 118 - 161 ( 25 ) ltekqfmdmneelncgm leignevcvd atvtpgsscytisasyg seq id no : 153 3 . 07 187 - 226 ( 9 ) lnlaelqttyncdalqv ddticvs ieilngdtcgflenafq seq id no : 154 3 . 85 236 - 279 tnntemeianegvkcdn lpigrmmcvw bacillus # ypbe htvqkketlyrismkyy seq id no : 155 9 . 45 191 - 136 240 l47648 subtilis ksrtgeekiraynhlng ndvytgqvldip citrobacter # eae ytlktgesvaqlsksqg seq id no : 156 8 . 59 65 - 113 936 q07591 fruendii isvpviwslnkhlysse semmkaspgqqiilp escherichia # eae ytlktgetvadlsksqd seq id no : 157 5 . 65 65 - 113 934 p43261 necessary coli inlstiwslnkhlysse for close semmkaapqqiilp ( intimate ) attachment of bacteria micrococcus # rpf ivvksgdslwtlaneye seq id no : 158 3 . 85 171 - 218 220 z96935 bacterial luteus veggwtalyeankgavs cytokine daaviyvgqelvl bacillus # ynea ievqqgdtlwsiadq seq id no : 159 3 . 81 40 - 90 105 z73234 subtilis vadtkkindkndfiewv adknqlqtsdiqpgdel vip streptococcus # ytvkygdtlstiaeamg seq id no : 160 4 . 23 47 - 103 393 u09352 pyogenes idvhvlgdinhianidl ifpdtiltanynqhgqa ttlt bacillus # xkdp ytvkkgdtlwdiagrfy seq id no : 161 11 . 23 176 - 234 235 p54335 subtilis gnstqwrkiwnanktam ikrskrnirqpghwifp gqklkip bacillus # yqbp ytvkkgdtlwdiagrfy seq id no : 161 11 . 23 177 - 234 235 g1225954 subtilis gnstqwrkiwnanktam ikrskrnirqpghwifp gqklkip bacillus # ytvkkgdtlwdlagkfy seq id no : 162 10 . 75 161 - 218 219 p45932 subtilis gdstkwrkiwkvnkka nikrskrnirqpghwif pgqklkip a ) proteins listed were obtained by a homology search in the swissprot , pir , and genbank databases with the repeats of acma using the blast program . b )* genes encoding cell wall hydrolases . # proteins containing repeats that are longer than the con - sensus sequence . c ) the number of aa residues between the repeats are given between brackets . d ) number of aa of the primary translation product . e ) genbank accession number . consensus repeat yxvkxgdtlxxiaxxxxxxxxxlxxxnxxlxxxxxixxgqxixvx ( seq id no : 163 ) h ir esv ls i i l l i l i v v v l [ 0164 ] table 4 calculated pi &# 39 ; s of individual repeat sequences of the acma and acmd protein anchors . acma anchor domain acmd anchor domain calculated calculated repeat pi repeat pi a1 9 . 75 d1 4 . 15 a2 9 . 81 d2 3 . 78 a3 10 . 02 d3 4 . 15 a1a2a3 10 . 03 d1d2d3 3 . 85 [ 0165 ] table 5 hybrid protein anchors composed of different acma and acmd repeat sequences and their calculated pi &# 39 ; s . composition of hybrids calculated acma - repeat sequence acmd - repeat sequence pi a1a2a3 — 10 . 03 a1a2 d1 9 . 53 a1a2a3 d1d2d3 8 . 66 a1 d2 8 . 45 a3 d1d2 7 . 39 a1a2 d1d2d3 6 . 08 a3 d1d2d3 5 . 07 a1 d1d2d3 4 . 37 — d1d2d3 3 . 85 tyr thr val lys ser gly asp thr leu trp gly ile ser gln arg tyr lys thr ser ile ala gln leu lys ser trp asn his leu ser ser asp gly ser pro ile ala ser ile lys ala trp asn his leu ser ser asp tyr lys val gln glu gly asp ser leu ser ala ile ala ala gln tyr tyr thr val lys ser gly asp ser leu tyr ser ile ala glu gln tyr gly met thr val ser ser leu met ser ala asn gly ile tyr asp val tyr val val lys gln gly asp thr leu ser gly ile ala ser asn trp gly thr asn trp gln glu leu ala arg gln asn ser leu ser asn pro asn met ile tyr ala gly gln val ile ser phe thr tyr thr val gln ser gly asp asn leu ser ser ile ala ile leu leu tyr ile val lys gln gly asp thr leu ser gly ile ala ser asn leu gly thr asn trp gln glu leu ala arg gln asn ser leu ser asn pro tyr thr val gln ser gly asp asn leu ser ser ile ala arg arg leu tyr thr val lys ser gly asp thr leu asn lys ile ala ala gln tyr tyr thr val lys ser gly asp thr leu asn lys ile ala ala gln tyr gly val thr val ala asn leu arg ser trp asn gly ile ser gly asp tyr thr ile lys ser gly asp thr leu asn lys ile ala ala gln tyr tyr thr ile lys ser gly asp thr leu asn lys ile ser ala gln phe gly val ser val ala asn leu arg ser trp asn gly ile lys gly asp his thr val lys ser gly asp ser leu trp gly leu ser met gln tyr tyr thr val lys ser gly asp ser val trp gly ile ser his ser phe gly ile thr met ala gln leu ile glu trp asn asn ile lys asn asn tyr thr val lys ser gly asp ser val trp lys ile ala asn asp his tyr thr val lys ala gly glu ser val trp ser val ser asn lys phe tyr thr val lys ala gly glu ser val trp gly val ala asn lys his his ile thr met asp gln leu ile glu trp asn asn ile lys asn asn tyr thr val lys ala gly glu ser val trp gly val ala asp ser his val val val glu ala gly asp thr leu trp gly ile ala gln ser lys his ala val lys ser gly asp thr ile trp ala leu ser val lys tyr val val val glu ala gly asp thr leu trp gly ile ala gln ser lys his asn val lys ser gly asp thr ile trp ala leu ser val lys tyr val val val glu ala gly asp thr leu trp gly ile ala gln asp lys tyr thr val lys ser gly asp thr ile trp ala leu ser ser lys tyr tyr thr val lys ser gly asp thr leu ser lys ile ala thr thr phe val val val glu ala gly asp thr leu trp gly ile ala gln asp asn his thr val lys ser gly asp thr ile trp ala leu ser val lys tyr tyr thr val lys ser gly asp thr leu gly lys ile ala ser thr phe val val val glu ala gly asp thr leu trp gly ile ala gln ser lys his thr val lys ser gly asp thr ile trp ala leu ser val lys tyr tyr thr val lys ser gly asp ser leu ser lys ile ala asn thr phe tyr lys val lys ser gly asp thr ile trp ala leu ser val lys tyr gly val pro val gln lys leu ile glu trp asn asn leu ser ser ser tyr lys val gln asn gly asp ser leu gly lys ile ala ser leu phe ile thr ile tyr ala gly gln glu leu ser val lys his lys val thr lys asn gln thr leu tyr ala ile ser arg glu tyr tyr lys val asn lys gly asp thr met phe leu ile ala tyr leu ala gly ile asp val lys glu leu ala ala leu asn asn leu ser glu pro tyr lys val arg lys gly asp thr met phe leu ile ala tyr ile ser gly met asp ile lys glu leu ala thr leu asn asn met ser glu pro his val val leu pro lys glu thr leu ser ser ile ala lys arg tyr tyr ile val arg arg gly asp thr leu tyr ser ile ala phe arg phe tyr ile val lys pro gly asp thr leu phe ser ile ala phe arg tyr gly trp asp tyr lys glu leu ala ala arg asn gly ile pro ala pro ile met val arg gln gly asp thr val thr val leu ala arg arg phe his gln val lys glu gly glu ser leu trp gln ile ser gln ala phe his val val lys ala gly glu thr ile asp ser ile ala ala gln tyr tyr lys val lys lys gly asp ser leu trp lys ile ala lys glu tyr tyr arg val lys arg gly asp ser leu ile lys ile ala lys lys phe tyr arg val arg arg gly asp thr leu ile lys ile ala lys arg phe tyr thr ile gln pro gly asp thr phe trp ala ile ala gln arg arg tyr thr ile gln pro gly asp thr phe trp ala ile ala gln arg arg asn met ile lys pro gly gln glu leu val val asp his lys val lys ser gly glu ser leu thr thr ile ala glu lys tyr gln ile thr leu gln ser leu lys asp ala asn pro gly val val pro ile val tyr arg glu gly asp ser leu ser lys ile ala lys lys tyr tyr val val gln asp gly asp ser leu trp leu ile ala lys arg phe val val val lys lys gly asp phe leu glu arg ile ala arg ala asn his thr thr val ala lys leu met gln ile asn asp leu thr thr thr tyr ile val gln glu gly asp ser pro trp thr ile ala leu arg asn val ile val lys lys gly asp phe leu glu arg ile ala arg ser asn tyr val val lys glu gly asp ser pro trp ala ile ala leu ser asn his ile val lys gln gly glu thr leu ser lys ile ala ser lys tyr his thr val arg ser asn glu ser leu tyr asp ile ser gln gln tyr tyr thr val gln ser gly asp thr leu gly ala ile ala ala lys tyr gly thr thr tyr gln lys leu ala ser leu asn gly ile gly ser pro tyr thr val arg ser gly asp thr leu gly ala ile ala ala lys tyr gly thr thr tyr gln lys leu ala ser leu asn gly ile gly ser pro tyr lys val gln lys asn asp thr leu tyr ser ile ser leu asn phe tyr thr val lys lys gly asp thr leu asp ala ile ala lys arg phe phe thr thr ala thr phe ile lys glu ala asn gln leu lys ser tyr his val val lys arg gly glu thr leu trp ser ile ala asn gln tyr gly leu gly val asp thr leu lys ala his asn arg ala his ser ala his val val gln gln gly asp thr leu trp ser leu ala lys arg tyr his val ile ala lys gly glu thr leu phe ser leu ser arg arg tyr his lys ile lys pro gly glu thr leu ser his val ala ala arg tyr gln ile thr ser glu thr leu ile ser phe asn glu ile lys asp val tyr lys val val lys gly asp thr leu phe ser ile ala ile lys tyr tyr thr ala lys glu gly asp thr ile glu ser ile ser lys leu val gly leu ser gln glu glu ile ile ala trp asn asp leu arg ser lys tyr met val arg lys gly asp ser leu ser lys leu ser gln asp phe his tyr val lys arg gly glu thr leu gly arg ile ala tyr ile tyr his ser val ala val gly glu thr leu tyr ser ile ala arg his tyr asn gly trp gln ser gly ile ala thr ile asn leu leu asp ser pro tyr thr val val ser gly asp ser trp trp lys ile ala gln arg asn gly leu ser met tyr thr leu ala ser gln asn gly lys ser ile tyr ile lys val lys lys gly asp thr leu trp asp leu ser arg lys tyr glu ile thr ser gln gln leu ile arg met asn ser leu ala leu asp met val lys gln gly asp thr leu ser ala ile ala ser gln tyr arg tyr asp val lys arg gly asp thr leu thr ser ile ala arg gln phe ile lys val lys ser gly asp ser leu trp lys leu ala gln thr tyr tyr thr val lys ser gly asp ser leu trp leu ile ala asn glu phe lys met thr val gln glu leu lys lys leu asn gly leu ser ser asp tyr lys val gln leu gly asp ser leu trp lys ile ala asn lys val tyr thr val lys ser gly asp ser leu trp lys ile ala asn asn tyr tyr thr val lys ser gly asp ser leu trp val ile ala gln lys phe asn val thr ala gln gln ile arg glu lys asn asn leu lys thr asp ile lys val lys ser gly asp ser leu trp lys leu ser arg gln tyr tyr thr val ala tyr gly asp ser leu trp met ile ala lys asn his tyr thr val lys leu gly asp ser leu trp lys ile ala asn ser leu asn met thr val ala glu leu lys thr leu asn gly leu thr ser asp tyr lys val lys ala gly asp ser leu trp lys ile ala asn arg leu gly val thr val gln ser ile arg asp lys asn asn leu ser ser asp ile thr val gln lys gly asp thr leu trp gly ile ser gln lys asn tyr thr ile lys ala gly asp thr leu ser lys ile ala gln lys phe his his val thr pro gly glu thr leu ser ile ile ala ser lys tyr asn val ser leu gln gln leu met glu leu asn his phe lys ser asp tyr his val lys lys gly asp thr leu ser gly ile ala ala ser his gly ala ser val lys thr leu gln ser ile asn his ile thr asp pro his ile val gln lys gly asp ser leu trp lys ile ala glu lys tyr his ile val gly pro gly asp ser leu phe ser ile gly arg arg tyr gly ala ser val asp gln ile arg gly val asn gly leu asp glu thr tyr gln val lys gln gly asp thr leu asn ser ile ala ala asp phe tyr val val lys gln gly asp thr leu thr ser ile ala arg ala phe gly val thr val ala gln leu gln glu trp asn asn ile glu asp pro tyr lys val lys ser gly asp asn leu thr lys ile ala lys lys his his lys val lys ser gly asp thr leu ser lys ile ala val asp asn lys thr thr val ser arg leu met ser leu asn pro glu ile thr asn pro asn his ile lys val gly gln thr ile arg leu ser his val val lys lys gly asp thr leu ser glu ile ala lys lys ile tyr thr ile thr gln gly asp ser leu trp gln ile ala gln asn lys tyr thr val lys lys gly asp thr leu phe tyr ile ala trp ile thr gly asn asp phe arg asp leu ala gln arg asn ser ile ser ala pro gly val ser thr lys asp leu gln gln trp asn lys leu arg gly ser tyr arg val arg lys gly asp ser leu ser ser ile ala lys arg his gly val asn ile lys asp val met arg trp asn ser asp thr ala asn gly thr ser val lys glu leu ala arg leu asn gly ile ser pro pro tyr thr val lys lys gly asp thr leu phe tyr ile ala trp ile thr tyr thr val gly asn arg asp thr leu thr ser val ala ala arg phe arg lys val lys asn gly asp thr leu asn lys leu ala ile lys tyr gln asp phe met ala leu ser lys val lys ile pro tyr thr ile thr glu thr asp thr leu glu arg val ala ala ser his asp cys thr val gly glu leu met lys leu asn lys met ala ser arg thr glu ile lys ser gly asp ser cys trp asn ile ala ser asn ala lys ile ser val glu arg leu gln gln leu asn lys gly met lys cys leu lys leu lys ala glu asp thr cys phe lys ile trp ser ser gln lys leu ser glu arg gln phe leu gly met asn glu gly met asp cys his lys ile gln lys gly asp thr cys phe lys ile trp thr thr asn lys ile ser glu lys gln phe arg asn leu asn lys gly leu asp cys leu lys ile lys glu gly asp thr cys tyr asn ile trp thr ser gln lys ile ser glu gln glu phe met glu leu asn lys gly leu asp cys tyr arg phe lys lys gly asp thr cys tyr lys ile trp thr ser his lys met ser glu lys gln phe arg ala leu asn arg gly ile asp cys ile thr val lys pro gly asp thr cys phe ser ile trp thr ser gln lys met thr gln gln gln phe met asp ile asn pro glu leu asp cys val lys ile asn pro gly asp thr cys phe asn ile trp thr ser gln val gln ile asn pro gly asp thr cys phe lys ile trp ser ala gln lys leu thr glu gln gln phe met glu leu asn lys gly leu asp cys thr glu val lys glu gly asp thr cys phe lys ile trp ser ala his lys ile thr glu gln gln phe met glu met asn arg gly leu asp cys ile lys val lys glu gly asp thr cys phe lys ile trp ser ala gln lys met thr glu gln gln phe met glu met asn arg gly leu asp cys ala thr ile thr pro gly asn thr cys phe asn ile ser val ala tyr gly ile asn leu thr asp leu gln lys thr tyr asp cys lys ala leu ile glu val ile lys gly asp thr cys trp phe leu glu asn ala phe lys thr asn gln thr glu met glu arg ala asn glu gly val lys cys asp asn leu pro ile gly arg met met cys val trp his thr ile lys ser gly asp thr cys trp lys 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