Patent Application: US-47281206-A

Abstract:
the invention relates to recombinant polypeptides and peptides and particularly to the polypeptide containing in its polypeptidic chain the following amino acid sequence : the one extending from the extremity constituted by nucleotide at position to the extremity constituted by nucleotide at position represented in fig . 4 a and fig . 4 b . the polypeptides and peptides of the invention can be used for the diagnostic of tuberculosis , and can also be part of the active principle in the preparation of vaccine against tuberculosis .

Description:
screening of the λqt11 m . tuberculosis recombinant dna library with anti - 32 - kda antiserum a λ . gt11 recombinant library constructed from genomic dna of m . tuberculosis ( erdman strain ), was obtained from r . young ( 35 ). screening was performed as described ( 14 , 35 ) with some modifications hereafter mentioned . λgt11 infected e . coli y1090 ( 10 5 pfu per 150 mm plate ) were seeded on nzym plates ( gibco ) ( 16 ) and incubated at 42 ° c . for 24 hrs . to induce expression of the β - galactosidase - fusion proteins the plates were overlaid with isopropyl β - d - thiogalactoside ( iptg )- saturated filters ( hybond c extra , amersham ), and incubated for 2 hrs at 37 ° c . screening was done with a polyclonal rabbit anti - 32 - kda antiserum . said polyclonal antiserum rabbit anti - 32 - kda antiserum was obtained by raising antiserum against the p 32 m . bovis bcg ( strain 1173p2 — institut pasteur paris ) as follows : 400 μg ( purified p 32 protein of m . bovis bcg ) per ml physiological saline were mixed with one volume of incomplete freund &# 39 ; s adjuvant . the material was homogenized and injected intradermally in 50 μl doses , delivered at 10 sites in the back of the rabbits , at 0 , 4 , 7 and 8 weeks ( adjuvant was replaced by the diluent for the last injection ). one week later , the rabbits were bled and the sera tested for antibody level before being distributed in aliquots and stored at − 80 ° c . the polyclonal rabbit anti - 32 - kda antiserum was pre - absorbed on e . coli lysate ( 14 ) and used at a final dilution of 1 : 300 . a secondary alkaline - phosphatase anti - rabbit igg conjugate ( promega ), diluted at 1 : 5000 was used to detect the β - galactosidase fusion proteins . for color development nitro blue tetrazolium ( nbt ) and 5 - bromo - 4 - chloro - 3 - indolyl phosphate ( bcip ) were used . reactive areas on the filter turned deep purple within 30 min . usually three consecutive purification steps were performed to obtain pure clones . iptg , bcip and nbt were from promega corp . ( madison wis .). plaque screening by hybridization for obtaining the secondary clones by1 , by2 and by5 hereafter defined the procedure used was as described by maniatis et al . ( 14 ). colonies of e . coli y1089 were lysogenized with appropriate λgt11 recombinants as described by hyunh et al . ( 14 ). single colonies of lysogenized e . coli y1089 were inoculated into lb medium and grown to an optical density of 0 . 5 at 600 nm at 30 ° c . after a heat shock at 45 ° c . for 20 min ., the production of β - galactosidase - fusion protein was induced by the addition of iptg to a final concentration of 10 mm . incubation was continued for 60 min at 37 ° c . and cells were quickly harvested by centrifugation . cells were concentrated 50 times in buffer ( 10 mm tris ph 8 . 0 , 2 mm edta ) and rapidly frozen into liquid nitrogen . the samples were lysed by thawing and treated with 100 μg / ml dnase i in ecori restriction buffer , for 5 - 10 minutes at 37 ° c . after sds - page electrophoresis , recombinant lysogen proteins were blotted onto nitrocellulose membranes ( hybond c , amersham ) as described by towbin et al . ( 29 ). the expression of mycobacterial antigens , fused to β - galactosidase in e . coli y1089 was visualized by the binding of a polyclonal rabbit anti - 32 - kda antiserum ( 1 : 1000 ) obtained as described in the above paragraph “ screening of the λgt11 m . tuberculosis recombinant dna library with anti - 32 - kda antiserum ” and using a monoclonal anti - β - galactosidase antibody ( promega ). a secondary alkaline - phosphatase anti - rabbit igg conjugate ( promega ) diluted at 1 : 5000 , was used to detect the fusion proteins . the use of these various antibodies enables to detect the β - galactosidase fusion protein . this reaction is due to the m . tuberculosis protein because of the fact that non fused β - galactosidase is also present on the same gel and is not recognized by the serum from tuberculous patients . in order to identify selective recognition of recombinant fusion proteins by human tuberculous sera , nitrocellulose sheets were incubated overnight with these sera ( 1 : 50 ) ( after blocking aspecific protein binding sites ). the human tuberculous sera were selected for their reactivity ( high or low ) against the purified 32 - kda antigen of m . bovis bcg tested in a dot blot assay as previously described ( 31 ). reactive areas on the nitrocellulose sheets were revealed by incubation with peroxidase conjugated goat anti - human igg antibody ( dakopatts , copenhagen , denmark ) ( 1 : 200 ) for 4 hrs and after repeated washings color reaction was developed by adding peroxidase substrate ( α - chloronaphtol ) ( bio - rad ) in the presence of peroxidase and hydrogen peroxide . initial identification of m . tuberculosis dna inserts in purified λgt11 clones was performed by ecori restriction . after digestion , the excised inserts were run on agarose gels and submitted to southern hybridization . probes were labeled with α 32 p - dctp by random priming ( 10 ). other restriction sites were located by single and double digestions of recombinant λgt11 phage dna or their subcloned ecori fragments by hindiii , psti , kpni , acci and sphl . sequence analysis was done by the primer extension dideoxy termination method of sanger et al . ( 25 ) after subcloning of specific fragments in bluescribe - m13 ( 6 ) or in mp10 and mp11 m13 vectors ( methods in enzymology , vol . 101 , 1983 , p . 20 - 89 , joachim messing , new m13 vectors for cloning , academic press ). sequence analysis was greatly hampered by the high gc content of the m . tuberculosis dna ( 65 %). sequencing reactions were therefore performed with several dna polymerases : t7 dna polymerase (“ sequenase ” usb ), klenow fragment of dna polymerase i ( amersham ) and in some cases with amv reverse transcriptase ( super rt , anglian biotechnology ltd .) and sometimes with ditp instead of dgtp . several oligodeoxynucleotides were synthesized and used to focus ambiguous regions of the sequence . the sequencing strategy is summarized in fig2 . in order to trace possible artefactual frameshifts in some ambiguous regions , a special program was used to define the most probable open reading frame in sequences containing a high proportion of gc ( 3 ). several regions particularly prone to sequencing artefacts were confirmed or corrected by chemical sequencing ( 18 ). for this purpose , fragments were subcloned in the chemical sequencing vector pgv462 ( 21 ) and analysed as described previously . selected restriction fragments of about 250 - 350 bp were isolated , made blunt - ended by treatment with either klenow polymerase or mung bean nuclease , and subcloned in the smai or hincii site of pgv462 . both strands of the inserted dna were sequenced by single - end labeling at tth 111i or bsteii ( 32 ) and a modified chemical degradation strategy ( 33 ). routine computer aided analysis of the nucleic acid and deduced amino acid sequences were performed with the lgbc program from bellon ( 2 ). homology searches used the fasta programs from pearson and lipman ( 23 ) and the protein identification resource ( pir ) from the national biomedical research fundation — washington ( nbrf ) ( nbrf / pir data bank ), release 16 ( march 1988 ). screening of the λgt11lm , m . tuberculosis recombinant dna library with polyclonal anti - 32 - kda antiserum ten filters representing 1 . 5 . times . 10 6 plaques were probed with a polyclonal rabbit anti - 32 - kda antiserum ( 8 ). following purification , six independent positive clones were obtained . ecori restriction analysis of these 6 purified λgt11 recombinant clones dna , ( fig1 a ) revealed 4 different types of insert . clone 15 had an insert with a total length of 3 . 8 kb with two additional internal ecori sites resulting in three dna fragments of 1 . 8 kb , 1 . 5 kb and 0 . 5 kb . the dna insert of clone 16 was 1 . 7 kb long . clones 17 and 19 had a dna insert of almost identical length being 2 . 7 kb and 2 . 8 kb respectively . finally , clone 23 ( not shown ) and clone 24 both contained an insert of 4 kb with one additional ecori restriction site giving two fragments of 2 . 3 kb and 1 . 7 kb . southern analysis ( data not shown ) showed that the dna inserts of clones 15 , 16 , 19 and the small fragment ( 1 . 7 kb ) of clone 24 only hybridized with themselves whereas clone 17 ( 2 . 7 kb ) hybridized with itself but also equally well with the 2 . 3 kb dna fragment of clone 24 . clones 15 , 16 and 19 are thus distinct and unrelated to the 17 , 23 , 24 group . this interpretation was further confirmed by analysis of crude lysates of e . coli y1089 lysogenized with the appropriate λgt11 recombinants and induced with iptg . western blot analysis ( fig1 b ), showed no fusion protein , either mature or incomplete , reactive with the polyclonal anti - 32 - kda antiserum in cells expressing clones 15 , 16 and 19 . clones 15 , 16 and 19 , were thus considered as false positives and were not further studied . on the contrary , cells lysogenized with clone 23 and 24 produced an immunoreactive fusion protein containing about 10 kda of the 32 - kda protein . clone 17 finally expressed a fusion protein of which the foreign polypeptide part is about 25 kda long . the restriction endonuclease maps of the 2 . 3 kb insert of clone 24 and of the 2 . 7 kb fragment of clone 17 ( fig2 ) allowed us to align and orient the two inserts suggesting that the latter corresponds to a ± 0 . 5 kb 5 ′ extension of the first . as clone 17 was incomplete , the same λgt11 recombinant m . tuberculosis dna library was screened by hybridization with a 120 bp ecori - kpni restriction fragment corresponding to the very 5 ′ end of the dna insert of clone 17 ( previously subcloned in a blue scribe vector commercialized by vector cloning systems ( stratagene cloning system ) ( fig2 ). three 5 ′- extended clones by1 , by2 and by5 were isolated , analyzed by restriction and aligned . the largest insert , by5 contained the information for the entire coding region ( see below ) flanked by 3 . 1 kb upstream and 1 . 1 kb downstream ( fig2 ). the 1358 base pairs nucleotide sequence derived from the various λkg11 overlapping clones is represented in fig3 a and fig3 b . the dna sequence contains a 1059 base pair open reading frame starting at position 183 and ending with a tag codon at position 1242 . it occurs that the nh 2 - terminal amino - acid sequence , ( phe - ser - arg - pro - gly - leu - pro - val - gl - u - tyr - leu - gln - val - pro - ser - pro - ser - met - gly - arg - asp - ile - lys - val - gln - phe - gln -- ser - gly - gly - ala - asn ; seq id no : 33 ) which can be located within this open reading frame from the nucleotide sequence beginning with a ttt codon at position 360 corresponds to the same nh 2 - terminal amino acid sequence of the mpb 59 antigen except for the amino acids at position 20 , 21 , 31 , which are respectively gly , cys and asn in the mpb 59 ( 34 ). therefore , the dna region upstream of this sequence is expected to encode a signal peptide required for the secretion of a protein of 32 - kda . the mature protein thus presumably consists of 295 amino acid residues from the n - terminal phe ( ttt codon ) to the c - terminal ala ( gcc codon ) ( fig5 ). six atg codons were found to precede the ttt at position 360 in the same reading frame . usage of any of these atgs in the same reading frame would lead to the synthesis of signal peptides of 29 , 42 , 47 , 49 , 55 and 59 residues . the hydropathy pattern coding sequence of the protein of 32 - kda of the invention and that of the antigen α of bcg ( 17 ) were determined by the method of kyte and doolittle ( 15 ). the nonapeptide profiles are shown in fig6 . besides the initial hydrophobic signal peptide region , several hydrophilic domains could be identified . it is interesting to note that the overall hydrophilicity pattern of the protein of 32 - kda of the invention is comparable to that of the bcg antigen α . for both proteins , a domain of highest hydrophilicity could be identified between amino acid residues 200 and 250 . matsuo et al . ( 17 ) recently published the sequence of a 1095 nucleotide cloned dna corresponding to the gene coding for the antigen a of bcg . the 978 bp coding region of m . bovis antigen α as revised in infection and immunity , vol . 58 , p . 550 - 556 , 1990 , and 1017 bp coding regions of the protein of 32 - kda of the invention show a 77 . 5 % homology , in an aligned region of 942 bp . at the amino acid level both precursor protein sequences share 75 . 6 % identical residues . in addition , 17 . 6 % of the amino acids correspond to evolutionary conserved replacements as defined in the algorithm used for the comparison ( pam250 matrix , ref23 ). fig7 shows sequence divergences in the n - terminal of the signal peptide . the amino terminal sequence — 32 amino acids — of both mature proteins is identical except for position 31 . fig8 shows that serum samples from tuberculous patients when immunoblotted with a crude e . coli extract expressing clone 17 distinctly react with the 140 kda fusion protein ( lanes 4 to 6 ) contain the protein of 32 - kda of the invention , but not with unfused β - galactosidase expressed in a parallel extract ( lanes 10 to 12 ). serum samples from two negative controls selected as responding very little to the purified protein of 32 - kda of the invention does neither recognize the 140 kda fused protein containing the protein of 32 - kda of the invention , nor the unfused β - galactosidase ( lanes 2 , 3 and 8 and 9 ). the 140 k - da fused protein and the unfused β - galactosidase were easily localized reacting with the anti - β - galactosidase monoclonal antibody ( lanes 1 to 7 ). the invention has enabled to prepare a dna region coding particularly for a protein of 32 - kda ( cf . fig5 ); said dna region containing an open reading frame of 338 codons ( stop codon non included ). at position 220 a ttt encoding the first amino acid of the mature protein is followed by the 295 triplets coding for the mature protein of 32 - kda . the size of this open reading frame , the immunoreactivity of the derived fusion proteins , the presence of a signal peptide and , especially , the identification within this gene of a nh 2 - terminal region highly homologous to that found in the mpb 59 antigen ( 31 / 32 amino acids homology ) and in the bcg antigen α ( 31 / 32 amino acids homology ) ( see fig7 ), strongly suggest that the dna fragment described contains the complete cistron encoding the protein of 32 - kda secreted by m . tuberculosis , and which had never been so far identified in a non ambiguous way . six atg codons were found to precede this ttt at position 220 in the same reading frame . usage of any of these atgs in the same reading frame would lead to the synthesis of signal peptides of 43 , 48 , 50 , 56 or 60 residues . among these various possibilities , initiation is more likely to take place either at atg 91 or atg 52 because both are preceded by a plausible e . coli - like promoter and a shine - dalgarno motif . if initiation takes place at atg 91 , the corresponding signal peptide would code for a rather long peptide signal of 43 residues . this length however is not uncommon among secreted proteins from gram positive bacteria ( 5 ). it would be preceded by a typical e . coli shine - dalgarno motif ( 4 / 6 residues homologous to aggagg ) at a suitable distance . if initiation takes place at atg 52 , the corresponding signal peptide would code for a peptide signal of 56 residues but would have a less stringent shine - dalgarno ribosome binding site sequence . the region encompassing the : translation termination triplet was particularly sensitive to secondary structure effects which lead to so - called compressions on the sequencing gels . in front of the tag termination codon at position 1105 , 22 out of 23 residues are g - c base pairs , of which 9 are g &# 39 ; s . upstream atg 130 , a sequence resembling an e . coli promoter ( 11 ) comprising an hexanucleotide ( ttgaga ) ( homology 5 / 6 to ttgaca ) and a aagaat box ( homology 4 / 6 to tataat ) separated by 16 nucleotides was observed . upstream the potential initiating codon atg 91 , one could detect several sequences homologous to the e . coli “- 35 hexanucleotide box ”, followed by a sequence resembling a tataat box . among these , the most suggestive is illustrated on fig3 a and 3 b . it comprises a ttggcc at position 59 ( fig3 a and 3 b ) ( homology 4 / 6 to ttgaca ) separated by 14 nucleotides from a gataag ( homology 4 / 6 to tataat ). interestingly this putative promoter region shares no extensive sequence homology with the promoter region described for the bcg protein α - gene ( 17 ) nor with that described for the 65 kda protein gene ( 26 , 28 ). searching the nbrf data bank ( issue 16 . 0 ) any significant homology between the protein of 32 - kda of the invention and any other completely known protein sequence could not be detected . in particular no significant homology was observed between the 32 - kda protein and α and β subunits of the human fibronectin receptor ( 1 ). the nh 2 - terminal sequence of the 32 - kda protein of the invention is highly homologous — 29 / 32 amino acids — to that previously published for bcg mpb 59 antigen ( 34 ) and to that of bcg α - antigen — 31 / 32 amino acids —( matsuo , 17 ) and is identical in its first 6 amino acids with the 32 - kda protein of m . bovis bcg ( 9 ). however , the presumed initiating methionine precedes an additional 29 or 42 amino acid hydrophobic sequence which differs from the one of α - antigen ( cf . fig7 ), but displaying all the characteristics attributed to signal sequences of secreted polypeptides in prokaryotes ( 22 ). interestingly , no significant homology between the nucleic acid ( 1 - 1358 ) of the invention ( cf . fig3 a and 3 b ) and the dna of the antigen α of matsuo exists within their putative promoter regions . construction of a bacterial plasmid containing the entire coding sequence of the 32 - kda protein of m . tuberculosis in the previous example , in fig2 , the various overlapping λgt11 isolates covering the 32 - kda protein gene region from m . tuberculosis were described . several dna fragments were subcloned from these λgt11 phages in the blue scribe m13 + plasmid ( stratagene ). since none of these plasmids contained the entire coding sequence of the 32 - kda protein gene , a plasmid containing this sequence was reconstructed . 1 ) the plasmid bs - by5 - 800 obtained by subcloning hindiii fragments of by5 ( cf . fig2 ) into the blue scribe m13 + plasmid ( stratagene ), was digested with hindiii and a fragment of 800 bp was obtained and isolated from a 1 % agarose gel by electroelution . 2 ) the plasmid bs - 4 . 1 obtained by subcloning the 2 . 7 kb ecori insert from λgt11 - 17 ) into the blue scribe m13 + plasmid ( stratagene ) ( see fig2 of patent application ) was digested with hindiii and sphi and a fragment of 1500 bp was obtained and isolated from a 1 % agarose gel by electroelution . 3 ) blue scribe m13 + was digested with hindiii and sphi , and treated with calf intestine alkaline phosphatase ( special quality for molecular biology , boehringer mannheim ) as indicated by the manufacturer . 50 ng of the hindiii - sphi digested bsm13 + vector ( 3 ) 100 μl of dh5 α e . coli ( gibco brl ) were transformed with 10 μl of the ligation reaction ( step 2 ) and plated on iptg , x - gal ampicillin plates , as indicated by the manufacturer . about 70 white colonies were obtained . as the 800 bp fragment could have been inserted in both orientations , plasmidic dna from several clones were analyzed by digestion with psti in order to select one clone ( different from clone 11 ), characterized by the presence of 2 small fragments of 229 and 294 bp . this construction contains the hindiii - hindiii - sphi complex in the correct orientation . the plasmid containing this new construction was called : “ bs . bk . p 32 complet ”. the dna sequence coding for a polypeptide , or part of it , can be linked to a ribosome binding site which is part of the expression vector , or can be fused to the information of another protein or peptide already present on the expression vector . in the former case the information is expressed as such and hence devoid of any foreign sequences ( except maybe for the aminoterminal methionine which is not always removed by e . coli ). in the latter case the expressed protein is a hybrid or a fusion protein . the gene , coding for the polypeptide , and the expression vector are treated with the appropriate restriction enzyme ( s ) or manipulated otherwise as to create termini allowing ligation . the resulting recombinant vector is used to transform a host . the transformants are analyzed for the presence and proper orientation of the inserted gene . in addition , the cloning vector may be used to transform other strains of a chosen host . various methods and materials for preparing recombinant vectors , transforming them to host cells and expressing polypeptides and proteins are described by panayatatos , n ., in “ plasmids , a practical approach ( ed . k . g . hardy , irl press ) pp . 163 - 176 , by old and primrose , principals of gene manipulation ( 2d ed , 1981 ) and are well known by those skilled in the art . various cloning vectors may be utilized for expression . although a plasmid is preferable , the vector may be a bacteriophage or cosmid . the vector chosen should be compatible with the host cell chosen . moreover , the plasmid should have a phenotypic property that will enable the transformed host cells to be readily identified and separated from those which are not transformed . such selection genes can be a gene providing resistance to an antibiotic like for instance , tetracycline carbenicillin , kanamycin , chloramphenicol , streptomycin , etc . in order to express the coding sequence of a gene in e . coli the expression vector should also contain the necessary signals for transcription and translation . hence it should contain a promoter , synthetic or derived from a natural source , which is functional in e . coli . preferably , although usually not absolutely necessary , the promoter should be controllable by the manipulator . examples of widely used controllable promoters for expression in e . coli are the lac , the trp , the tac and the lambda pl and pr promoter . preferably , the expression vector should also contain a terminator of transcription functional in e . coli . examples of used terminators of transcription are the trp and the rrnb terminators . furthermore , the expression vector should contain a ribosome binding site , synthetic or from a natural source , allowing translation and hence expression of a downstream coding . sequence . moreover , when expression devoid of foreign sequences is desired , a unique restriction site , positioned in such a way that it allows ligation of the sequence directly to the initiation codon of the ribosome binding site , should be present . a suitable plasmid for performing this type of expression is pkk233 - 2 ( pharmacia ). this plasmid contains the trc promoter , the lac z ribosome binding site and the rmb transcription terminator . also suitable is plasmid pigri ( innogenetics , ghent , belgium ). this plasmid contains the tetracycline resistance gene and the origin of replication of pat 153 ( available from bioexcellence , biores b . v ., woerden , the netherlands ), the lambda pl promoter up to the mboii site in the 5 ′ untranslated region of the lambda n gene ( originating from ppl ( λ ); pharmacia ). downstream from the pl promoter , a synthetic sequence was introduced which encodes a “ two cistron ” translation casette whereby the stop codon of the first cistron ( being the first 25 amino acids of tnf , except for leu at position 1 which is converted to val ) is situated between the shine - dalgarno sequence and the initiation codon of the second ribosome binding site . the restriction and genetic map of pigri is represented in fig1 a . fig1 b and table 5 represent respectively the nucleic acid sequence and complete restriction site analysis of pigri . however , when expression as a hybrid protein is desired , then the expression vector should also contain the coding sequence of a peptide or polypeptide which is ( preferably highly ) expressed by this vector in the appropriate host . in this case the expression vector should contain a unique cleavage site for one or more restriction endonucleases downstream of the coding sequence . plasmids pex1 , 2 and 3 ( boehringer , mannheim ) and puex1 , 2 and 2 ( amersham ) are useful for this purpose . they contain an ampicillin resistance gene and the origin of replication of pbr322 ( bolivar at al . ( 1977 ) gene 2 , 95 - 113 ), the lac z gene fused at its 5 ′ end to the lambda pr promoter together with the coding sequence for the 9 first amino acids of its natural gene cro , and a multiple cloning site at the 3 ′ end of the lac z coding sequence allowing production of a beta galactosidase fused polypeptide . the puex vectors also contain the ci857 allele of the bacteriophage lambda ci repressor gene . also useful is plasmid pmtnf mph ( innogenetics ). it contains the tetracycline resistance gene and the origin of replication of pat 153 ( obtainable from bioexcellence , biores b . v ., woerden . the netherlands ), the lambda pl promoter up to the mboii site in the n gene 5 ′ untranslated region ( originating from ppl ( λ ); pharmacia ), followed by a synthetic ribosome binding site ( see sequence data ), and the information encoding the first 25 aa of mtnf ( except for the initial leu which is converted to val ). this sequence is , in turn , followed by a synthetic polylinker sequence which encodes six consecutive histidines followed by several proteolytic sites ( a formic acid , cnbr , kallikrein , and e . coli protease vii sensitive site , respectively ), each accessible via a different restriction enzyme which is unique for the plasmid ( smai , ncoi , bspmii and stui , respectively ; see restriction and genetic map , fig1 a ). downstream from the polylinker , several transcription terminators are present including the e . coli trp terminator ( synthetic ) and the rrnbt 1 t 2 ( originating from pkx223 - 3 ; pharmacia ). the total nucleic acid sequence of this plasmid is represented in fig1 b . table 6 gives a complete restriction site analysis of pmtnf mph . the presence of 6 successive histidines allows purification of the fusion protein by immobilized metal ion affinity chromatography ( imac ). after purification , the foreign part of the hybrid protein can be removed by a suitable protein cleavage method and the cleaved product can then be separated from the uncleaved molecules using the same imac based purification procedure . in all the above - mentioned plasmids where the lambda pl or pr promoter is used , the promoter is temperature - controlled by means of the expression of the lambda ci ts 857 allele which is either present on a defective prophage incorporated in the chromosome of the host ( k12δh , atcc n o 33767 ) or on a second compatible plasmid ( pacyc derivative ). only in the puex vectors is this ci allele present on the vector itself . it is to be understood that the plasmids presented above are exemplary and other plasmids or types of expression vectors maybe employed without departing from the spirit or scope of the present invention . if a bacteriophage or phagemid is used , instead of plasmid , it should have substantially the same characteristics used to select a plasmid as described above . fifteen μg of plasmid “ bs - bk - p 32 complet ” ( see example ii ) was digested with eclxi and bsteii ( boehringer , mannheim ) according to the conditions recommended by the supplier except that at least 3 units of enzyme were used per μg of dna . eclxi cuts at position 226 ( fig5 ) and bsteii at position 1136 , thus approaching very closely the start and stop codon of the mature p 32 antigen . this dna is hereafter called dna coding for the “ p 32 antigen fragment ”. the dna coding for the “ p 32 antigen fragment ” ( as defined above ) is subcloned in pigri ( see fig1 a ) for expression of a polypeptide devoid of any foreign sequences . to bring the atg codon of the expression vector in frame with the p 32 reading frame , an intermediary construct is made in pig2 ( for restriction and genetic map , see fig1 a ; dna sequences , see fig1 b ; complete restriction site analysis , see table 7 ). five μg of plasmid pig2 is digested with ncoi . its 5 ′ sticky ends are filled in prior to dephosphorylation . therefore , the dna was incubated in 40 μl nb buffer ( 0 . 05 m tris - cl ph 7 . 4 ; 10 mm mgcl 2 ; 0 . 05 % β - mercaptoethanol ) containing 0 . 5 mm of all four dxtp ( x = a , t , c , g ) and 2 μl of klenow fragment of e . coli dna polymerase i ( 5 u / μl , boehringer , mannheim ) for at least 3 h at 15 ° c . after blunting , the dna was once extracted with one volume of phenol equilibrated against 200 mm tris - cl ph 8 , twice with at least two volumes of diethylether and finally collected using the “ gene clean kit ™” ( biol101 ) as recommended by the supplier . the dna was then dephosphorylated at the 5 ′ ends in 30 μl of cip buffer ( 50 mm triscl ph 8 , 1 mm zncl 2 ) and 20 to 25 units of calf intestine phosphatase ( high concentration , boehringer , mannheim ). the mixture was incubated at 37 ° c . for 30 min , then egta ( ethyleneglycol bis ( β - aminoethylether )- n , n , n ′, n ′ tetraacetic acid ) ph 8 is added to a final concentration of 10 mm . the mixture was then extracted with phenol followed by diethylether as described above , and the dna was precipitated by addition of 1 / 10 volume of 3 m kac ( ac = ch 3 coo ) ph 4 . 8 and 2 volumes of ethanol followed by storage at − 20 ° c . for at least one hour . after centrifugation at 13000 rpm in a biofuge a ( hereaus ) for 5 min the pelleted dna was dissolved in h 2 o to a final concentration of 0 . 2 μg / μl . the eclxi - bsteii fragment , coding for the “ p 32 antigen fragment ” ( see above ) was electrophoresed on a 1 % agarose gel ( brl ) to separate it from the rest of the plasmid and was isolated from the gel by centrifugation over a millipore hvlp filter ( φ2 cm ) ( 2 min , 13000 rpm , biofuge at room temperature ) and extracted with tris equilibrated phenol followed by diethylether as described above . the dna was subsequently collected using the “ gene clean kit ™” ( bio101 ) as recommended by the supplier . after that , the 5 ′ sticky ends were blunted by treatment with the klenow fragment of e . coli dna polymerase i as described above and the dna was then again collected using the “ gene clean kit ™” in order to dissolve it in 7 μl of h 2 o . one μl of vector dna is added together with one μl of 10 × ligase buffer ( 0 . 5 m triscl ph 7 . 4 , 100 mm mgcl 2 , 5 mm atp , 50 mm dtt ( dithiothreitol )) and 1 μl of t4 dna ligase ( 1unit / μl , boehringer , mannheim ). ligation was performed for 6 h at 13 ° c . and 5 μl of the mixture is then used to transform strain dh1 ( lambda ) [ strain dh1 — atcc n o 33849 — lysogenized with wild type bacteriophage λ ] using standard transformation techniques as described for instance by maniatis et al . in “ molecular cloning , a laboratory manual ”, cold spring harbor laboratory ( 1982 ). individual transformants are grown and lysed for plasmid dna preparation using standard procedures ( experiments with gene fusions , cold spring harbor laboratory ( 1984 ) ( t . j . silhavy , h . l . berman and l . w . enquist , eds ) and the dna preparations are checked for the correct orientation of the gene within the plasmid by restriction enzyme analysis . a check for correct blunting is done by verifying the restoration of the ncoi site at the 5 ′ and 3 ′ end of the antigen coding sequence . one of the clones containing the p 32 antigen fragment in the correct orientation is kept for further work and designated pig 2 - mt32 . in this intermediary construct , the dna encoding the antigen is not in frame with the atg codon . however , it can now be moved as a ncoi fragment to another expression vector . 15 μg of pig 2 - mt32 is digested with ncoi . the ncoi fragment encoding the p 32 antigen is gel purified and blunted as described above . after purification , using “ gene clear kit tm ” it is dissolved in 7 μl of h 2 o . 5 μg of plasmid pigri is digested with ncoi , blunted and dephosphorylated as described above . after phenol extraction , followed by diethylether and ethanolprecipitation , the pellet is dissolved in h 2 o to a final concentration of 0 . 2 μg / μl . ligation of vector and “ antigen fragment ” dna is carried out as described above . the ligation mixture is then transformed into strain dh1 ( lambda ) and individual transformants are analysed for the correct orientation of the gene within the plasmid by restriction enzyme analysis . a check for correct blunting is done by verifying the creation of a new nsii site at the 5 ′ and 3 ′ ends of the antigen coding sequence . one of the clones containing the p 32 antigen fragment in the correct orientation is kept for further work and designated pigri . mt32 . fifteen μg of the plasmid pig2 mt32 ( see example iv ) was digested with the restriction enzyme ncoi ( boehringer , mannheim ), according to the conditions recommended by the supplier except that at least 3 units of enzyme were used per μg of dna . after digestion , the reaction mixture is extracted with phenol equilibrated against 200 mm triscl ph 8 , ( one volume ), twice with diethylether ( 2 volumes ) and precipitated by addition of 1 / 10 volume of 3 m kac ( ac ═ ch 3 coo ) ph 4 . 8 and 2 volumes of ethanol followed by storage at − 20 ° c . for at least one hour . after centrifugation for 5 minutes at 13000 rpm in a biofuge a ( hereaus ) the dna is electrophoresed on a 1 % agarose gel ( brl ). the dna coding for the “ p 32 antigen fragment ” as described above , is isolated by centrifugation over a millipore hvlp filter ( φ2 cm ) ( 2 minutes , 13000 rpm , biofuge at room temperature ) and extracted one with triscl equilibrated phenol and twice with diethylether . the dna is subsequently collected using “ gene clean kit ™” ( bio 101 ) and dissolved in 7 μl of h 2 o . the 5 ′ overhanging ends of the dna fragment generated by digestion with ncoi were filled in by incubating the dna in 40 μl nb buffer ( 0 . 05 m tris - hcl , ph 7 . 4 ; 10 mm mgcl 2 ; 0 . 05 % β - mercaptoethanol ) containing 0 . 5 mm of all four dxtps ( x = a , t , c , g ) and 2 μl of klenow fragment of e . coli dna polymerase i ( 5 units / μl boehringer mannheim ) for at least 3 h at 15 ° c . after blunting , the dna was extracted with phenol , followed by diethylether , and collected using a “ gene clean kit ™” as described above . five μg of plasmid pmtnf mph is digested with stui , subsequently extracted with phenol , followed by diethylether , and precipitated as described above . the restriction digest is verified by electrophoresis of a 0 . 5 μg sample on an analytical 1 . 2 % agarose gel . the plasmid dna is then desphosphorylated at the 5 ′ ends to prevent self - ligation in 30 μl of cip buffer ( 50 mm triscl ph 8 , 1 mm znc12 ) and 20 to 25 units of calf intestine phosphatase ( high concentration , boehringer mannheim ). the mixture is incubated at 37 ° c . for 30 minutes , then egta ( ethyleneglycol bis ( β - aminoethylether )- n , n , n ′, n ′ tetraacetic acid ) ph8 is added to a final concentration of 10 mm . the mixture is extracted with phenol followed by diethylether and the dna is precipitated as described above . the precipitate is pelleted by centrifugation in a biofuge a ( hereaus ) at 13000 rpm for 10 min at 4 ° c . and the pellet is dissolved in h 2 o to a final dna concentration of 0 . 2 μg / μl . one μl of this vector dna is mixed with the 7 μl solution containing the dna fragment coding for the “ p32antigen fragment ” ( as defined above ) and 1 μl 10 × ligase buffer ( 0 . 5 m triscl ph7 . 4 , 100 mm mgcl2 , 5 mm atp , 50 mm dtt ( dithiothreitol )) plus 1 μl t4 dna ligase ( 1 unit / μl , boehringer mannheim ) is added . the mixture is incubated at 13 ° c . for 6 hours and 5 μl of the mixture is then used for transformation into strain dh1 ( lambda ) using standard transformation techniques are described by for instance maniatis et al . in “ molecular cloning , a laboratory manual ”, cold spring harbor laboratory ( 1982 ). individual transformants are grown and then lysed for plasmid dna preparation using standard procedures ( experiments with gene fusions , cold spring harbor laboratory 1984 ( t . j . silhavy , m . l . berman and l . w . enquist eds .)) and are checked for the correct orientation of the gene within the plasmid by restriction enzyme analysis . one of the clones containing the dna sequence encoding the antigen fragment in the correct orientation was retained for further work and designated pmtnf - mph - mt32 . it encodes all information of the p 32 antigen starting from position + 4 in the amino acid sequence ( see fig5 ). the amino acid sequence of the total fusion protein is represented in fig1 . dna of pmtnf - mph - mt32 is transformed into e . coli strain k12δh ( atcc 33767 ) using standard transformation procedures except that the growth temperature of the cultures is reduced to 28 ° c . and the heat shock temperature to 34 ° c . a culture of k12 . delta . h harboring pmtnf - mph - mt32 , grown overnight in luria broth at 28 ° c . with vigorous shaking in the presence of 10 μg / ml tetracycline , is inoculated into fresh luria broth containing tetracycline ( 10 μg / ml ) and grown to an optical density at 600 nanometers of 0 . 2 in the same conditions as for the overnight culture . when the optical density at 600 nanometers has reached 0 . 2 half of the culture is shifted to 42 ° c . to induce expression while the other half remains at 28 ° c . as a control . at several time intervals aliquots are taken which are extracted with one volume of phenol equilibrated against m9 salts ( 0 . 1 % ammonium chloride , 0 . 3 % potassium dihydrogenium phosphate , 1 . 5 % disodium hydrogenium phosphate , 12 molecules of water ) and 1 % sds . at the same time , the optical density ( 600 nm ) of the culture is checked . the proteins are precipitated from the phenol phase by addition of two volumes of acetone and storage overnight at − 20 ° c . the precipitate is pelleted ( biofuge a , 5 min ., 13000 rpm , room temperature ) dried at the air , dissolved in a volume of laemmli ( nature ( 1970 ) 227 : 680 ) sample buffer (+ β - mercapto ethanol ) according to the optical density and boiled for 3 min . samples are then run on a sds polyacrylamide gel ( 15 %) according to laemmli ( 1970 ). temperature induction of mtnf - his 6 - p 32 is monitored by both coomassie brilliant blue ( cbb ) staining and immunoblotting . cbb staining is performed by immersing the gel in a 1 / 10 diluted cbb staining solution ( 0 . 5 g cbb - r250 ( serva ) in 90 ml methanol : h 2 o ( 1 : 1 v / v ) and 10 ml glacial acetic acid ) and left for about one hour on a gently rotating platform . after destaining for a few hours in destaining solution ( 30 % methanol , 7 % glacial acetic acid ) protein bands are visualised and can be scanned with a densitometer ( ultroscan xl enhanced laser densitometer , lkb ). for immunoblotting the proteins are blotted onto hybond c membranes ( amersham ) as described by townbin et al ( 1979 ). after blotting , proteins on the membrane are temporarily visualised with ponceau s ( serva ) and the position of the molecular weight markers is indicated . the stain is then removed by washing in h 2 o . a specific protein binding sites are blocked by incubating the blots in 10 % non - fat dried milk for about 1 hour on a gently rotating platform . after washing twice with nt buffer ( 25 mm tris - hcl , ph 8 . 0 ; 150 mm nacl ) blots are incubated with polyclonal rabbit anti - 32 - kda antiserum ( 1 : 1000 ), obtained as described in example i (“ screening of the λgt11 m . tuberculosis recombinant dna library with anti - 32 - kda antiserum ”) in the presence of e . coli lysate or with monoclonal anti - htnf - antibody which crossreacts with mtnf ( innogenetics , n o 17f5d10 ) for at least 2 hours on a rotating platform . after washing twice with nt buffer + 0 . 02 % triton . x . 100 , blots are incubated for at least 1 hour with the secondary antiserum alkaline phosphatase - conjugated swine anti - rabbit immunoglobulins ( 1 / 500 ; prosan ) in the first case , and alkaline phosphatase conjugated rabbit anti - mouse immunoglobulins ( 1 / 500 ; sigma ) in the second case . blots are washed again twice with nt buffer + 0 . 02 % triton x100 and visualisation is then performed with nitro blue tetrazolium ( nbt ) and 5 - bromo - 4 - chloro - 3 - indolyl - phosphate ( bcip ) from promega using conditions recommended by the supplier . upon induction of k12 . delta . h cells containing pmtnf - mph - mt32 , a clearly visible band of about 35 - kda appears on cbb stained gels , already one hour after start of induction ( fig1 a ). this band , corresponding to roughly 25 % of total protein contents of the cell , reacts strongly with anti - 32 - kda and anti - mtnf antisera on immunoblots ( fig1 b ). however , this band represents a cleavage product of the original fusion protein , since a minor band , around 37 kda , is also visible on immunoblots , reacting specifically with both antisera as well . this suggests that extensive cleavage of the recombinant mtnf - his 6 - p 32 takes place about 2 - 3 kda from its carboxyterminal end . purification of recombinant antigen on immobilized metal ion affinity chromatography ( imac ) the hybrid protein mtnf - his 6 - p 32 ( amino acid sequence , see fig1 ) expressed by k12δh cells containing pmtnf . mph . mt32 , is especially designed to facilitate purification by imac , since the 6 successive histidines in the polylinker sequence bring about a strong affinity for metal ions ( hochuli et al , 1988 ). 12 l of e . coli cells k12ah containing plasmid pmtnf - mph - mt32 were grown in luria broth containing tetracycline ( 10 μg / ml ) at 28 ° c . to an optical density ( 600 nm ) of 0 . 2 and then induced by shifting the temperature to 42 ° c . after 3 hours of induction , cells were harvested by centrifugation ( beckman , j a 10 rotor , 7 , 500 rpm , 10 min ). the cell paste was resuspended in lysis buffer ( 10 mm kcl , 10 mm tris - hcl ph 6 . 8 , 5 mm edta ) to a final concentration of 50 % ( w / v ) cells . ε - nh 2 - capronic acid and dithiotreitol ( dtt ) were added to a final concentration of resp . 20 mm and 1 mm , to prevent proteolytic degradation . this concentrated cell suspension was stored overnight at − 70 ° c . cells were lysed by passing them three times through a french press ( slm - aminco ) at a working pressure of 800 - 1000 psi . during and after lysis , cells were kept systematically on ice . the cell lysate was cleared by centrifugation ( beckman , j a 20 , 18 , 000 rpm , 20 min , 4 ° c .). the supernatant ( sn ) was carefully taken off and the pellet , containing membranes and inclusion bodies , was kept for further work since preliminary experiments had shown that the protein was mainly localised in the membrane fraction . 7 m guanidinium hydrochloride ( guhcl , marketed by icn ) in 100 mm phosphate buffer ph 7 . 2 was added to the pellet volume to a final concentration of 6 m guhcl . the pellet was resuspended and extracted in a bounce tissue homogenizer ( 10 cycles ). after clearing ( beckman , j a 20 , 18 , 000 rpm , 20 min , 4 ° c . ), about 100 ml of supernatant was collected (= extract 1 ) and the removing pellet was extracted again as described above (= extract 2 , 40 ml ). the different fractions ( sn , ex1 , ex2 ) were analysed on sds - page ( laemmli , nature 1970 ; 227 : 680 ) together with control samples of the induced culture . scanning of the gel revealed that the recombinant protein makes up roughly 25 % of the total protein content of the induced cell culture . after fractionation most of the protein was found back in the extracts . no difference was noticed between reducing and non - reducing conditions ( plus and minus β - mercaptoethanol ). b . preparation of the ni ++ ida ( imino diacetic acid ) column : 5 ml of the chelating gel , chelating sepharose 6b ( pharmacia ) is washed extensively with water to remove the ethanol in which it is stored and then packed in a “ econo - column ” ( 1 × 10 cm , biorad ). the top of the column is connected with the incoming fluid ( sample , buffer , etc ) while the end goes to the uv 280 detector via a peristaltic jump . fractions are collected using a fraction collector and , when appropriate , ph of the fractions is measured manually . the column is loaded with ni ++ ( 6 ml nicl 2 . 6h 2 o ; 5 μg / μl ) and equilibrated with starting buffer ( 6 m guanidinium hydrochloride , 100 mm phosphate buffer , ph 7 . 2 ). after having applied the sample , the column is washed extensively with starting buffer to remove unbound material . to elute the bound material , 2 different elution procedures are feasible : to regenerate the column , which has to be done after every 2 - 3 runs , 20 ml ( about 5 column volumes ) of the following solutions are pumped successively through the column : 0 . 05 m edta - 0 . 5 m nacl 0 . 1 m naoh h 2 o 6 ml nicl 2 . 6 h 2 o ( 5 mg / ml ). after equilibrating with starting buffer the column is ready to use again . all buffers contained 6 m guanidinium hydrochloride throughout the chromatography . the column was developed at a flow rate of 0 . 5 ml / min at ambient temperature . fractions of 2 ml were collected and , when appropriate , further analysed by sds - page and immunoblotting . gels were stained with coomassie brilliant blue r250 and silver stain , as described by ansorge ( 1985 ). immunoblotting was carried out as described in example i . the primary antiserum used was either polyclonal anti - 32 kda - antiserum ( 1 / 1000 ) obtained as described in example i (“ screening of the λgt11 m . tuberculosis recombinant dna library with anti - 32 kda - antiserum ”) or anti - e . coli - immunoglobulins ( 1 / 500 ; prosan ), or monoclonal anti - htnf - antibody which cross - reacts with mtnf ( innogenetics , n o 17f5d 10 ). the secondary antiserum was alkaline phosphatase conjugated swine anti - rabbit immunoglobulins ( 1 / 500 , prosan ), or alkaline phosphatase conjugated rabbit - anti - mouse immunoglobulins ( 1 / 500 , sigma ). after applying 3 ml of extract 1 ( od 280 = 32 . 0 ) and extensively washing with solution a , the column is equilibrated with solution b and then developed with a linear ph gradient from 7 . 2 to 4 . 2 ( 25 ml of solution b and 25 ml of solution c were mixed in a gradient former ). the elution profile is shown in fig1 . from sds - page analysis ( coomassie and silverstain ) it was clear that most of the originally bound recombinant protein was eluted in the fractions between ph 5 . 3 and 4 . 7 . screening of these fractions on immunoblot with anti - 32 - kda and the 17f5d10 monoclonal antibody showed that , together with the intact recombinant protein , also some degradation products and higher aggregation forms of the protein were present , although in much lower amount . blotting with anti - e . coli antibody revealed that these fractions ( ph 5 . 3 - 4 . 7 ) still contained immunodetectable contaminating e . coli proteins ( 75 , 65 , 43 , 35 and 31 kda bands ) and lipopolysaccharides . sample application and washing was carried out as in c1 , except that after washing , no equilibration was necessary with 6 m guhcl 25 mm phosphate . the column was first developed with a linear gradient of imidazol going from 0 to 50 mm ( 25 ml of solution a and 25 ml of solution b were mixed in a gradient former ) followed by a step elution to 100 mm imidazol ( solution c ). during the linear gradient , proteins were gradually eluted in a broad smear , while the step to 100 mm gave rise to a clear peak ( fig1 ). sds - page analysis of the fractions revealed that in the first part of the linear gradient ( fr 1 - 24 ) most contaminating e . coli proteins were washed out , while the latter part of the gradient ( fr 25 - 50 ) and the 100 mm peak contained more than 90 % of the recombinant protein . as in c1 , these fractions showed , besides a major band of intact recombinant protein , some minor bands of degradation and aggregation products . however , in this case , the region below 24 - kda seemed nearly devoid of protein bands , which suggests that less degradation products co - elute with the intact protein . also , the same contaminating e . coli proteins were detected by immunoblotting , as in c1 , although the 31 - kda band seems less intense and even absent in some fractions . in a second stage , we developed the column with a step gradient of increasing imidazol concentrations . after having applied the sample and washed the column , 2 column volumes ( about 8 ml ) of the following solutions were brought successively onto the column solution d , e , f and finally 4 column volumes of solution c . the stepgradient resulted in a more concentrated elution profile ( fig1 ) which makes it more suitable for scaling up purposes . in conclusion , the mtnf - his 6 - p 32 protein has been purified to at least 90 % by imac . further purification can be achieved through a combination of the following purification steps : imac on chelating superose ( pharmacia ) ion exchange chromatography ( anion or cation ) reversed phase chromatography gel filtration chromatography immunoaffinity chromatography elution from polyacrylamide gel . these chromatographic methods are commonly used for protein purification . the plasmids of fig1 b , 11 b and 12 b are new . 1 . abou - 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