Patent Application: US-98152797-A

Abstract:
heterologous protein secretion from gram - positive bacteria , in particular from bacilli has , with few exceptions , met with little success . incompatibility of the heterologous proteins with the protein secretion machinery of the host is the main cause of this effect . this limiting factor for the production of heterologous proteins in commercially significant concentrations from bacillus subtilis is removed by overexpressing the bacillus subtilis protein ftsy or ftsy protein in combination with overexpression of other members of the bacterial signal recognition particle . said gene is overexpressed in bacillus host cells expressing a heterologous protein which then shows an increased amount of the heterologous protein secreted in the surrounding medium .

Description:
there is now growing evidence that poor expression and / or secretion is caused by incorrect folding of the heterologous protein in the host cell . the cause of this effect may be the incompatibility of the host cell &# 39 ; s chaperone - like proteins of the regular secretory pathway and the heterologous protein . as a result the newly synthesized heterologous proteins will be recognized very inefficiently and in this way become a rate - limiting step in the translocation process . this will be even more pronounced if the heterologous protein is overexpressed . one possibility to overcome this problem is to express heterologous chaperone - like proteins which are homologous to the heterologous protein which is to be secreted . expression of e . coli secb in b . subtilis has shown to facilitate secretion of the secb - dependent maltose - binding protein of e . coli ( collier , 1994 ). this option is probable not applicable when the heterologous protein and secretion factor are from a more phylogenetic distant organism . in this way the host cell &# 39 ; s regular secretion machinery could become incompatible with the heterologous chaperone - like protein itself , leaving the same effect : extreme low secretion efficiency . another possibility , part of this invention , is to overexpress one or more of thee host cell &# 39 ; s chaperone - like proteins , preferably the srp - like chaperone - like proteins and so increase the availability of these chaperone - like proteins for the heterologous protein . because homologues of seca ( sadaie et al . 1991 ), sece ( jeong et al . 1993 ), secy ( su et al . 1990 ), and lep ( van dijl et al . 1992 ) have been identified in b . subtilis , it is suggested that signal peptide - dependent protein secretion in b . subtilis utilizes a sec - pathway that is similar to that of e . coli . so far secb , which is considered to be the major chaperone in e . coli , seems to be the only chaperone which has a direct binding affinity for seca and so contributes to the accurate targeting of the preprotein - secb complex to the membrane bound translocase . the secb protein is needed for only a subset of the envelope proteins so secb independent proteins will enter the sec - pathway with the aid of helper proteins like groel / groes , dnak / dnaj or other proteins like srp . in eukaryotic organism srp mainly is responsible for the translocation across the er membrane . recently more evidence has become available of the existence of an srp mediated secretion route in bacteria . because the eukaryotic pathway has probably evolved from the bacteria it is thinkable that said proteins are also dependent on this pathway when said proteins are expressed in bacteria like bacillus . thus optimisation of this particularly pathway in bacillus will be more profitable for heterologous ( eukaryotic ) proteins secretion than the optimisation of the well known sec - pathway . this invention relates to the cloning of the bacillus ftsy gene and its effect after ( over )- expression , alone or in combination with other members of the bacterial srp , upon heterologous proteins . for the cloning of b . subtillis ftsy degenerate primers were synthesized making use of the existing homology boxes between the srα homologues of different organism ( fig2 a ). after an inversed pcr reaction using a 110 bp fragment , which was derived from a nested pcr reaction , as template a 4 kb fragment could be detected . sequencing results ( fig2 b ) revealed an open reading frame of 329 amino acids . this protein shared 48 . 2 % amino acid identity and 65 % similarity with the ftsy gene of e . coli . as will be shown in the examples , the hybridizing experiments originally lead to an unwanted result , i . e . a smear of indistinct bands . surprisingly , we were able by cutting out a region around the expected size of the amplified fragment and applying pcr to that region again in resolving this smear into a group of distinct bands . unfortunately hardly any band was seen at the expected size of the fragment that should have been amplified . however , in a third round of amplification we were nevertheless able to obtain a fragment which could be used further . phb201 is capable of autonomous replication in both e . coli ( high - copy ) and bacillus ( low - copy ) strains . this plasmid confers resistance to the antibiotics chloramphenicol and erythromycin in e . coli and bacillus . further the plasmid carries a cat86 :: lacz fusion gene preceded by the strong lactococcus lactis promoter 59 which also act as strong promoter in b . subtilis ( van der vossen et al . 1987 ). by replacing the sali / ecori fragment of this plasmid by a synthetic dna fragment ( seq id no : 1 ) the cat86 :: lacz was deleted and an unique ndei restriction site introduced overlapping the translation initiation site generating phbnde ( fig1 a ). this allows us to express the chaperone genes directly downstream the strong lactococcal promoter 59 without creating fusion proteins as would be with the original phb201 vector ( fig1 b ). molecular cloning of bacillus subtilis dna fragments homologous with the human srα gene a set of three polymerase chain reactions ( pcr ) were performed as follows . chromosomal dna of b . subtilis 168 was used as template in a first pcr reaction with degenerate primers ab4229 ( seq id no : 2 ) and ab4230 ( seq id no : 3 ). after 30 cycles and an annealing temperature of 40 ° c . the amplified dna was fractionised by electrophoresis on a 2 % metaphor ( fmc bioproducts ) agarose gel . the results showed a smear of ill resoluted bands . dna fragments ranging in size from 220 bp to 300 bp were purified from the agarose gel with a qiaquick gel extraction column ( qiagen ). 1 / 50 of these isolated fragments were used in a second pcr with degenerate primers ab4229 and ab 4241 ( seq id no : 4 ) using the same reaction conditions as in the first pcr . the result of this pcr showed a number of district bands , however , a band of the expected size (± 120 bp ) was hardly visible . fragments of ± 120 bp were isolated from the agarose gel as above and used in a third pcr with the same primers and conditions as were used for the second pcr . the resulting single fragment was isolated , purified and after treatment with t4 polynucleotide kinase ligated into dephosphorylated puc18 linearized with smai . after electroporation to e . coli jm109 , selection on iptg / x - gal plates , the dna from six white colonies were used for automated sequencing . within all six isolates an open reading frame ( orf ) could be detected . this orf showed over 70 % similarity with an alignment of homologs from several other organisms ( fig2 b ). sequencing of unknown dna sequences adjacent to a short stretch of known sequence by using a labelled internal ftsy fragment derived from example 2 we could detect a single 4 kb psti band in a hybridisation experiment . none of the attempts to clone this fragment directly into puc were successful , indicating that cloning of this fragment could be lethal in e . coli . an inversed pcr ( ipcr ) was used for determination of the sequence . a total psti digest of b . subtilis 168 chromosomal dna was used as template in the ipcr with primers ab5356 ( seq id no : 5 ) and ab5357 ( seq id no : 6 ). the resulting fragment was used directly for automated sequencing making use of the same primers . the sequence of the rest of the psti chromosomal dna fragment located upstream of the primer ab5357 and downstream of primer ab5357 was determined by automated sequencing making use of newly developed primers while the sequence was unveiled . the total 4370 bp dna sequence of the psti fragment is shown in fig3 . analysis of the sequence showed the presence of several open reading frames ( orfs ), including the one for ftsy . when comparing the overall structure of the srα like proteins and srp54 - like proteins a common domain is evident which comprises gtp binding boxes ( the g - domain , see fig4 ). also from this figure it is clear that the b . subtilis ftsy protein contains only a very short n - terminal domain , in contrast to the eukaryotic and e . coli homologues . since the n - terminal domain in those organisms serves as a membrane anchor it is possible that in b . subtilis ftsy functions with a different mechanism , and is possibly more chaperone - like in its action , although it may still be membrane - bound . analysis of the sequence showed the presence of several more orf &# 39 ; s , including a truncated orf showing homology to the ffh protein , and a truncated orf showing homology to several dna segregation proteins like the yeast protein smc1 . the chromosomal organisation of the genes in the psti fragment is shown in fig5 . the effects of depletion of ftsy in bacillus on the processing and / or secretion of heterologous proteins were studied by placing the chromosomal ftsy gene under control of the inducible spac promoter ( yansura et . al ). for this the n - terminal part of the ftsy gene was cloned into the multiple cloning site of pdg148 directly downstream the spac promoter . the spac - ftsy - penp - laci fragment from the resulting pdgftsy ′ plasmid was recloned into pppneo2 making the final integration construct pnsftsy ′ ( fig6 ). pnsftsy ′ is capable of autonomous replication in e . coli but not in bacillus . it confers resistance to the antibiotic ampicillin which can be used for selection in e . coli and neomycin for selection in bacillus . integration of pnsftsy ′ into the ftsy locus results in a truncated copy of ftsy ( ftsy ′) under control of the authentic promoter and an intact copy of ftsy under control of the spac promoter . neomycin resistant pnsftsy ′ integrants could be selected after a protoplast transformation of b . subtilis 168 . integrants growing in a medium with 0 . 5 mm iptg showed growth characteristics comparable to b . subtilis host lacking the integrated plasmid . the growth rate of integrants did not decrease after incubation in the absence of iptg , nor did the cell morphology . effects of depletion of ftsy on protein translocation were examined by fermentation experiments using hosts expressing heterologous proteins . a pub110 like vector containing regulatory sequences of the b . licheniformis or b . amyloliquefaciens α - amylase gene was used for the expression of heterologous proteins . some of the heterologous proteins were produced to slightly higher levels in cells cultured in the presence of iptg , however in the absence of iptg the production of the heterologous proteins was not completely abolished . these effects were unexpected since in e . coli depletion of ftsy has a profound effect on the cell morphology and growth rate . therefore it is possible that there is ftsy formation despite the absence of iptg , indicating transcriptional read through . it should be possible to correct this by insertion of a strong terminator signal upstream of the spac promoter in the integration construct . to confirm that the absence of ftsy is hazardous to the cell attempts were made to disrupt the ftsy gene . since the experiments described in example 4 were unexpected in the sense that strain containing the ftsy gene under the control of the spac promoter were still viable , and showed no clear phenotype in the absence of the inducer iptg , we hypothesized that the construct we used was leaky so that even in the absence of iptg a small amount of ftsy would be produced . to eliminate the production of ftsy we tried to disrupt the ftsy gene by insertion of a neomycin resistance marker . we constructed a plasmid pbhdsptsyneo which harbours the 5 ′ and the 3 ′ end of the ftsy gene separated by the neomycin resistance gene in a vector unable to replicate in b . subtilis . plasmid pbhdsftsyneo was linearized by cutting with hpal and used to transform b . subtills 168 to neomycin resistance . this should lead to strains having the original ftsy gene replaced by the fsty :: neo construct via a double cross - over event ( see fig7 ). however , we were unable to select for neomycin resistant colonies using this linear dna , suggesting the possibility that disruption of the ftsy gene is lethal to the cell . we therefore repeated the transformation experiments with intact , uncut , plasmid dna . in this case integration of the plasmid into the ftsy gene can take place via a single cross - over event ( campbell type integration ), leading to neomycin resistant colonies which have both an intact and a disrupted copy of the ftsy gene present ( see fig8 ). we isolated integrant strains , one of which was shown to have the chromosomal organization represented in fig8 . since in this strain two copies of the 3 ′ end of the fsty gene are present recombination between them is possible , leading to excision of the plasmid sequences in between and formation of a strain containing only one copy of the ftsy gene , disrupted by the neomycin resistance marker . despite several attempts we were unable to isolate such recombinant strains , suggesting again that such strains are unviable . effect of ftsy overexpression on the location of precursors and mature heterologous proteins the effects of overexpression of ftsy in bacillus on the processing and / or secretion of heterologous proteins were studied by placing the complete ftsy gene under control of the constitutive p59 promoter in phbnde ( see example 1 ) resulting in the plasmid phbftsy . effects of overexpression of ftsy on protein translocation were examined by pulse - chase experiments using hosts expressing heterologous proteins . b . licheniformis t399 was transformed with plasmid plat - il3 containing the human interleukin - 3 ( h - il - 3 ) gene expressed from the b . licheniformis α - amylase promoter and provided with the b . licheniformis α - amylase signal sequence , or with the plasmid plp10 - ab containing the prochymosin gene under the same expression signals . the resulting strains t399il and t399chy were transformed with plasmid phbnftsy containing the ftsy gene . as a control also both strains t399il and t399chy were transformed with the vector phbnde . single colonies of all strains were inocculated in 5 ml of medium starvation medium s7 + ( including methionine and cysteine ) and grown at 37 ° c . overnight . aliquots of 200 μl were inocculated in 5 ml s7 − ( without methionine and cysteine ) medium and grown for another 5 hours . after growth to od = 0 . 3 - 0 . 7 a sample of 3 . 2 ml was centrifuged , washed with s7 − medium and resuspended into 3 . 2 ml fresh s7 − medium . the sample was incubated for 20 minutes at 37 ° c . and pulsed with 25μ ( ci l − [ 35 s ]- methionine (& gt ; 1000 ci / mmol ) per ml during 60 seconds at 37 ° c . then a chase was performed by addition of 50 μl ( 2 mg / ml ) l - methionine per ml . the chase was stopped at different time points ( 0 , 15 , 30 , and 60 seconds ) by mixing of 600 μl of the reaction with 600 μl ice cold 20 % tca , and incubation on ice for at least 30 minutes . the samples were centrifugated , and the supernatant was used directly for immuno precipitation , sds - polyacrylamide gel electrophoresis and autoradiography using standard protocols . the cell pellet was washed with 1 ml aceton , and dried . the cells were resuspended in 50 / l lysis buffer 10 mm tris ph8 , 25 mm mgcl , 200 mm nacl , 5 mg / ml lysozyme ) and incubated for 3 minutes at 37 ° c . after addition of 50 μl tes ( 20 mm tris , 2 mm edta , 2 % sds , ph8 ) the samples were boiled for 5 minutes . to the samples was added 900 μl of stdt ( 10 mm tris , 0 . 9 % nacl , 1 % triton , 0 . 5 % sodium deoxycholate , phs . 2 ), the mixture was incubated for 15 - 60 minutes on ice , and the debris was precipitated . the supernatant was used for immuno precipitation with antiserum raised against h - il - 3 or chymosin and subsequent sds - polyacrylamide gel electrophoresis and autoradiography using standard protocols . overexpression of ftsy increased the secretion of the mature form of interleukin - 3 and the processing of the precursor of prochymosin . the ffh gene encoding the b . subtilis homologue of the eukaryotic srp54 protein was cloned as a pcr fragment obtained using primers based on the published dna sequence ( honda et al , 1993 ). the gene was cloned into the vector phbnde ( see example 1 ) under the control of the strong lactococcal p59 promoter to form plasmid phbnffh . pulse - chase experiments were performed as described in example 6 with b . licheniformis strains containing both plasmid phbnffh and plasmid plat - il3 harbouring the human interleukin - 3 ( h - il - 3 ) gene under the expression and secretion signals of the b . licheniformis α - amylase gene . as is shown in fig9 the processing of h - il - 3 is very fast , as no precursor can be detected . the amount of mature h - il - 3 in the supernatant fraction is in all cases is higher in the samples obtained from the strain overproducing ffh compared to the strain containing only the vector plasmid . the scr gene encoding the scrna from b . subtilis was cloned as a pcr fragment using primers based on the published dna sequence ( struck et al , 1989 ) and following the approach ( including the same 5 ′ primer ) described by nakamura ( nakamura et al , 1992 ) introducing a hindiii site just upstream of the scr dna and a sphi site downstream of the terminator sequence . the p59 promoter was deleted from the vector phb210 by replacement of the alwni - smal fragment containing the origin of replication ( ori ) together with the p59 promoter by the alwni - pvuii fragment from vector pbr322 containing only the same ori . this new vector pbhk was used to exchange the ecori - pvuii fragment for the ecori - bamri ( blunted by t4 polymerase ) containing the spac - penp - laci cassette from pdg148 to contract the vector pbhspac . the pcr fragment containing the scr gene was digested with hindiii and sphi and ligated into pbhspac digested with the same restriction enzymes to construct plasmid pbhsscr . this way the sir gene was placed under the control of the spac promoter . pulse - chase experiments were performed as described in example 6 with b . licheniformis is strains containing both plasmid pbhsscr and plasmid plat - il3 harbouring the human interleukin - 3 ( h - il - 3 ) gene under the expression and secretion signals of the b . licheniformis α - amylase gene . also in this case the processing of h - il - 3 was very fast , as no precursor could be detected . however , the amount of mature h - il - 3 in the supernatant fraction was in all cases higher in the samples obtained from the strain overproducing scrna compared to the strain containing only the vector plasmid , or with plasmid pbhsscr in the absence of the inducer iptg . effects of simultaneously overexpressed signal recognition particle components on the secretion of heterologous proteins the effects described in examples 6 , 7 and 8 were even more pronounced when more than one of the components of the bacterial signal recognition particle were simultaneously expressed . for this purpose the ftsy gene was expressed from the iptg inducible promoter located in the chromosome as described in example 4 by addition of 3 mm iptg , while the components ffh or scrna were expressed from their phe210 derived vectors described in examples 7 and 8 above . ausubel et al . 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