Patent Application: US-61269690-A

Abstract:
the invention relates to the construction of protease - deficient escherichia coli hosts which when combined with an expression system are useful for the production of proteolytically sensitive polypeptides . the invention also includes examples of particular mutant escherichia the united states government may have certain rights in the present invention pursuant to the terms of grant no . cbt - 8657471 awarded by the national science foundation .

Description:
the bacterial strains and plasmids used are listed in table i . bacteriophage t4 gene 2 mutant ( n51am ) was provided as a gift from e . goldberg ( tufts medical school , dept . of microbiology ). cultures were grown in lb medium ( difco ) supplemented with 0 . 2 % glucose and the appropriate concentrations of antibiotics . m9 medium was supplemented with 0 . 2 % casein amino acid hydrolysate , 0 . 2 % glucose and the appropriate antibiotics . w salts medium has been described ( 17 ) and consists of 10 . 5 g k 2 hpo 4 , 4 . 5 g kh 2 po 4 , 0 . 102 g mgso 4 . 7h 2 o per liter of solution containing 0 . 2 % l - glutamine , 0 . 2 % glucose , and 0 . 2 % ( nh 4 ) 2 so 4 . ampicillin , tetracycline , kanamycin and chloramphenicol were added to the growth medium as required at 50 μg / ml , 25 μg / ml , 50 μg / ml and 20 μg / ml final concentration respectively . mutant e . coli deficient in one or more genes encoding a protease have been deposited with the american type culture collection ( atcc ), 12301 parklawn drive , rockville , md . 20852 u . s . a . on oct . 10 . 1990 . the bacteria are identified as escherichia coli sf120 ( atcc 55099 ), escherchia coli sg103 ( atcc 55100 ) and escherchia coli sf110 ( atcc 55101 ) deficient respectively in ompt , degp and ptr genes ( atcc 55099 ), ptr gene ( atcc 55100 ), and ompt and degp genes ( atcc 55101 ). table i__________________________________________________________________________bacterial strains and plasmids . strain or plasmid genotype or characteristics source or reference__________________________________________________________________________straind301 rp487 recd1903 δ ( lacizya - u169 ) 23jc7729 k - 12 recb21 sbcb15 his327 leu ( am ) trpe9829 lac str321 thiks272 f . sup .- δlacx74 gale galk thi rpsl ( stra ) 14 δphoa ( pvuii ) ks474 ks272 degp41 ( δpsti - kan . sup . r ) 28sf100 ks272 δompt 16sf101 d301 ptr32 :: ωcat . sup . rsf103 ks272 ptr32 :: ωcat . sup . rsf110 ks272 δompt degp41 ( δpsti - kan . sup . r ) 16sf112 ka272 δompt ptr32 :: ωcat . sup . rsf115 ks272 ptr32 :: ωcat . sup . r degp41 ( δpsti - kan . sup . r ) sf120 ks272 ptr32 :: ωcat . sup . r degp41 ( δpsti - kan . sup . r ) δomptplasmidpacyc184 4 . 2 kbp medium copy number plasmid ; 29 cat . sup . r tet . sup . rpcdk3 pbr325 derivative carrying a 19 kbp 30 bamh i fragment mapping ( thya - arga ). pcs1 puc19 derivative carrying a 3 . 2 kbp pvul 16 spa - bla fragment from pfb3 ; amp . sup . r . pfb3 9 . 86 kbp pbr322 derivative carrying 15 spa - bla ; amp . sup . r kan . sup . r . pfb5 pcs1 derivative carrying an 8 kbp sal i fragment encoding ptr ; amp . sup . r . pfb6 pfb5 derivative ( ptr32 :: ωcat . sup . 4 ); amp . sup . r__________________________________________________________________________ cat . sup . r . restriction and dna modifying enzymes were purchased from boehringer - mannheim , new england biolabs and promega . all recombinant dna procedures were performed according to maniatis et ai . ( 18 ) or ausubel et al . ( 19 ). oxidized insulin b - chain and penicillin g were obtained from sigma . all chemicals used were of biological grade . southern blots were performed essentially as described by ausubel et ai . ( 19 ). genomic dna was isolated as described ( 19 ), separated in 0 . 8 % agarose gels and transferred overnight to nitrocellulose ( schleicher & amp ; schuell ba85 ). dna was crosslinked to nitrocellulose by baking for 2 hr at 80 ° c . in a vacuum oven . nonradioactive digoxigenin - 11 - dutp probes were prepared using the genius system ( boehringer mannheim ) according to the manufacturer &# 39 ; s instructions . cells were grown in labeling medium supplemented with 19 amino acids but no methionine ( 20 ) to mid - exponential phase ( od = 0 . 5 at 600 nm ). 100 μci of 35 s - met were added to the growth medium . after two to five minutes , 1 ml of 0 . 5 mg / ml cold methionine was added and a zero point taken immediately . at specified times after initiation of the chase , 1 ml samples were transferred to microfuge tubes kept on ice and containing 50 μl of 34 mg / ml chloramphenicol . the cells were centrifuged , washed with 1 ml of 10 mm tris hcl , ph 8 . 0 , resuspended in lysis buffer ( 10 mm tris hcl , ph 8 . 0 , 1 % sds , 1 mm edta ) and boiled for 3 minutes . immunoprecipitation was performed as described ( 16 ). autoradiograms were scanned with a prototype digital clayton video densitometer developed at the university of texas by larry poulson . 3 ml samples from cultures grown as specified were centrifuged at 8 , 000 × g for 8 min , the pellets were resuspended in 3 ml 50 mm potassium phosphate , ph 6 . 5 and disrupted by french pressing at 20 , 000 psi . the insoluble fraction was removed by centrifugation . penicillinase activities were measured by spectrophotometry at 240 nm as previously described ( 15 ) using a 0 . 5 g / l penicillin g solution in 50 mm potassium phosphate , ph 6 . 5 as a substrate . for insulin assays , cells were grown overnight in lb medium supplemented with 0 . 2 % glucose and the appropriate concentrations of antibiotics as required . osmotic fractions were collected by the method of nossal and heppel ( 21 ) and concentrated using amicon centricon - 10 microconcentrators . 30 μg of periplasmic proteins were mixed with 100 mm ammonium bicarbonate buffer , ph 8 . 4 , and exactly 250 μg of oxidized insulin b - chain was added . the final volume was 500 μl . samples were incubated for 3 . 5 hr at 37 ° c . 20 μl aliquots ( corresponding to an initial insulin concentration of 10 μg ) were boiled in loading buffer to stop the reaction and loaded onto a 22 % polyacrylamide gel . 10 μg of purified insulin was used as a control . sds - page was performed according to laemmli ( 22 ) in 22 % gels . protein concentrations were determined with the biorad protein assay using bovine serum albumin as a standard . two mutations were combined in a single strain using p1 transduction to transfer chromosomal dna from ompt mutant , escherichia coli strain ut4400 , into a degp mutant strain ks474 . strain ut4400 is a spontaneous mutant in which the entire ompt gene together with a sizable piece of adjacent dna has been deleted from the chromosome . strain ks474 contains a gene that confers resistance to the antibiotic kanamycin at the position where the degp gene is normally located . cells were selected for resistance to kanamycin and to colicin d ( resistance to this compound is encoded by one of the genes deleted from ompt mutant strain ut4400 ). in addition , the absence of ompt was tested by examining the outer membrane proteins by electrophoresis ( 16 ) the overall strategy used to construct ptr mutants is shown in fig1 . plasmid pcdk3 which carries a 19 kbp insert mapping the thya - arga region of the e . coli genome was digested with sal i and bamh i . the 8 kbp sal i fragment containing the ptr gene , was isolated by low melting point agarose . this dna fragment was ligated into the unique sal i site of plasmid pcs1 to yield plasmid pfb5 . plasmid pcs1 was itself generated by transferring the protein a - β - lactamase gene into a plasmid vector having a different origin of replication ( 16 ). pfb5 contains a unique cla i site , located approximately 1000 bp downstream from the start codon of the ptr gene . a functional chloramphenicol acetyl transferase ( cat ) gene was obtained in a 1 . 7 kbp hinc ii - xmn i fragment from plasmid pacyc184 . plasmid pfb5 was linearized with cla i , treated with klenow to generate blunt ends , and ligated to the 1 . 7 kbp dna fragment from pacyc184 . competent cells were transformed with the ligation mixture . transformants were selected on chloramphenicol plates and tested for ampicillin resistance . the structure of the resulting plasmid , pfb6 , was confirmed by restriction analysis . plasmid pfb6 was digested with sal i and the 9 . 7 kbp fragment was isolated . the chloramphenicol resistance insertional mutation in the ptr gene was transferred to the e . coli chromosome . e . coli strain d301 carries a tetracycline resistance insertional mutation in the recd gene that allows the cells to be transformed with linearized dna ( 23 ). competent d301 cells were transformed with about 1 . 5 μg of the 9 . 7 kbp linearized dna and plated on chloramphenicol plates . to ensure that no intact pfb6 had been cotransformed , the transformants were tested for ampicillin sensitivity . a chloramphenicol resistant ( cat r ), tetracycline resistant ( tet r ), ampicillin sensitive ( amp s ) colony , sf101 , was selected . the ptr mutation was designated ptr32 ::: ωcat r . since sf101 is a d301 derivative , it also contains the tetracycline resistance insertional mutation in its recd gene . the ptr mutation was transferred into bacterial strain ks272 by generalized p1 transduction . transductants containing the ptr32 :: ωcat r mutation were isolated on the basis of chloramphenicol resistance . these colonies were further selected for tetracycline sensitivity to ensure that they did not contain the recd mutation present in d301 . this process yielded an e . coli strain carrying a chloramphenicol resistance insertional mutation in the ptr gene as well as a functional recd gene , i . e . having an intact exonuclease v activity . the selected strain sf103 has been deposited with atcc , accession number 55100 . the correct insertion of the chloramphenicol cartridge was confirmed by southern blotting ( fig2 ). a hinc ii - xmn i chloramphenicol probe derived from plasmid pacyc184 and labeled with digoxigenin - 11 - dutp hybridized with hinc ii + cla i digested genomic dna from sf103 ( lane 2 ) but did not hybridize with dna from the parental strain ks272 . because protease iii is the only cell envelope protease which has been reported to rapidly degrade insulin , the absence of protease iii proteolytic activity in the ptr32 ::: ωcat r mutant sf103 was demonstrated as follows . strains ks272 and sf103 were grown in lb medium supplemented with glucose and the appropriate antibiotics for 24 hours . the periplasmic fraction of the cells was collected by the method of nossal and heppel ( 21 ) and concentrated by ultrafiltration . periplasmic proteins were incubated with oxidized insulin b - chain and resolved by sds - page as described in materials and methods . fig3 shows that no appreciable degradation was observed with sf103 ( lane 3 ). in contrast , no intact insulin could be detected after incubation with the osmotic shock fractions of the isogenic strains ks272 ( lane 2 ). therefore , the ptr32 :: ωcat r mutant sf103 does not possess the insulin degrading activity typical of strains containing an intact protease iii activity . finally , the presence of an intact exonuclease v activity in sf103 was demonstrated in the following manner . e . coli cells containing a defective exonuclease v enzyme ( e . g ., a mutation in one or more of the recb , recc and recd genes ) are fully permissive for bacteriophage t4 gene 2 mutants ( t4 2 - ). however , t4 2 - is unable to infect cells containing an intact exonuclease v activity . strains jc7729 , ks272 and sf103 were infected at different multiplicities of infection with t4 2 - ( amn51 ) as described ( 24 ). although t4 2 - formed numerous plaques on the recb strain jc7729 ( the titer of the phage was about 3 × 10 9 pfu / ml on jc7729 ), none were obtained on ks272 or sf103 , indicating the presence of a fully functional exonuclease v in these strains . furthermore , streaks of sf103 cells exposed to 1 . 1 mw of uv radiation per cm 2 for up to 100 seconds did not display the reduced viability typically observed in recb recc recd mutants . the ptr mutant sf103 grew to an optical density at 600 nm of about 1 . 8 in a variety of media ( e . g ., lb , m9 , w salts ). p1 generalized transduction was used to transfer the ptr32 :: ωcat r mutation from sf101 to a degp mutant ks474 . transductants were selected for chloramphenicol resistance and tetracycline sensitivity to confirm the presence of the ptr mutation and the absence of the recd mutation respectively . the resulting colonies were further tested for kanamycin resistance , indicating the presence of the degp mutation . the resulting double protease - deficient strain , sf115 , was tested for ( i ) proper insertion of the chloramphenicol resistance cartridge ( fig2 lane 3 ), ( ii ) absence of proteolytic activity against insulin , and ( iii ) presence of an intact exonuclease v activity , with a series of tests as described in example 2 . mutants in both ptr and ompt were constructed in a manner analogous to that used for the construction of the ptr degp double mutant sf115 , described in example 3 , except that p1 generalized transduction was used to transfer the ptr32 :: ωcat r mutation from sf101 to sf100 . the latter strain contains a deletion in the chromosome which includes the ompt gene ( 16 ). transductants were selected for chloramphenicol resistance and tetracycline sensitivity to confirm the presence of the ptr mutation and the absence of the recd mutation , respectively . the resulting double protease - deficient strain sf112 , was tested for ( i ) proper insertion of the chloramphenicol resistance cartridge , ( ii ) absence of proteolytic activity against insulin , and ( iii ) presence of an intact exonuclease v activity , as described in example 2 . generalized p1 transduction was used to transfer the ptr32 :: ωcat r mutation from strain sf101 obtained as described in example 2 into ompt degp mutants as obtained in example 1 . transductants were selected for chloramphenicol resistance and tetracycline sensitivity to confirm the presence of the ptr mutation and the absence of the recd mutation , respectively . the resulting colonies were further tested for kanamycin resistance , indicating the presence of the degp mutation . the selected triply protease - deficient mutant strain was designated sf120 and is on deposit with atcc , accession number 55099 . strain sf120 was tested for ( i ) proper insertion of the chloramphenicol resistance cartridge ( fig2 lane 4 ), ( ii ) absence of proteolytic activity against insulin ( fig3 lane 5 ), and ( iii ) presence of an intact exonuclease v activity , with a series of tests as described in example 2 . plasmid pfb3 encodes a hybrid protein constructed by in - frame fusion of the genes coding for staphylococcus aureus protein a and the e . coli enzyme tem β - lactamase ( fig4 ). β - lactamase is a small , monomeric enzyme involved in the hydrolysis of the β - lactam ring of many antibiotics such as penicillin , nitrocefin , and cephaloridine . β - lactamase activity against the substrate benzylpenicillin was determined using the spectrophotometric methods described in materials and methods . the protease - deficient mutants described in examples 1 to 6 were shown to augment the expression of protein a - β - lactamase as follows : strains ks272 , ks474 , sf103 , sf110 , sf115 and sf120 were transformed with the plasmid vector pfb3 . cultures were grown for 24 hours in lb medium and the total penicillinase activity was assayed as described in materials and methods . penicillinase specific activities of these strains are shown in table ii . table ii__________________________________________________________________________influence of different protease - deficient mutant strains on theexpression ofprotein a - β - lactamase from the plasmid vector pfb3 . total penicillinasestrain strain growth specific activity ( plasmid ) characteristics medium ( u / mg ) ± sd % increase__________________________________________________________________________ks272 ( pfb3 ) ompt . sup .+ degp . sup .+ ptr . sup .+ lb 15 . 2 + 2 . 9 -- ks474 ( pfb3 ) ompt . sup .+ degp ptr . sup .+ lb 47 . 9 ± 8 . 3 215sf100 ( pfb3 ) ompt degp . sup .+ ptr . sup .+ lb 28 . 2 ± 7 . 1 85sf103 ( pfb3 ) ompt . sup .+ degp . sup .+ ptr lb 32 . 6 ± 12 . 5 115sf110 ( pfb3 ) ompt degp ptr . sup .+ lb 87 . 9 ± 17 . 2 478sf115 ( pfb3 ) ompt . sup .+ degp ptr lb 77 . 5 ± 16 . 5 410sf120 ( pfb3 ) ompt degp ptr lb 86 . 9 ± 10 . 1 427__________________________________________________________________________ table ii indicates that a single mutation in the ptr gene ( strain sf103 described in example 2 ) increased the expression of protein a - β - lactamase by about 2 - fold compared to the parental strain ks272 . this improvement is comparable to that observed with the single ompt mutant sf100 ( lane 3 ). an approximate 3 - fold increase in expression is obtained with the single degp mutant ks474 . use of the double mutants sf110 ( ompt degp ) and sf115 ( ptr degp ) described in examples 1 and 3 , respectively , increases the expression of protein a - β - lactamase by more than 5 - fold relative to the parental strain ks272 . the triple mutant sf120 ( ptr degp ompt ) described in example 5 , also provided a 5 to 6 - fold increase in protein a - β - lactamase expression . enhanced expression levels of protein a - β - lactamase in the ompt degp double mutant sf110 were confirmed by radioactive pulse - chase experiments as described in materials and methods . fig5 shows that the half - life of the fusion protein was approximately 30 min in the degp single mutant strain ks474 , but was increased to about 100 minutes in the ompt degp double mutant strain sf110 . essentially no degradation of the fusion protein was evident in the double mutant for the first hour following the initiation of the chase , demonstrating that e . coli strains deficient in multiple proteases also provide enhanced levels of expression of protein a - β - lactamase in exponentially growing cells . similarly , no degradation of protein a - β - lactamase was observed in the ptr mutant sf103 for the first 30 minutes following the initiation of the chase ( fig6 ). effect of medium , ph and metal ions on protein a - β - lactamase expression in protease - deficient mutants strains sf110 ( ompt degp ) and sf120 ( ptr degp ompt ) were transformed with plasmid vector pfb3 and grown in m9 medium . total specific penicillinase activities were measured . the introduction of a ptr mutation in the degp , ompt background resulted in a reproducible 33 % increase in protein a - β - lactamase expression , as shown in table iii . table iii__________________________________________________________________________total penicillinase activities of double and triple protease - deficientmutantstransformed with the plasmid vector pfb3 . total penicillinasestrain strain growth specific activity ( plasmid ) characteristics medium ( u / mg ) ± sd % increase__________________________________________________________________________sf110 ( pfb3 ) ompt degp ptr . sup .+ m9 4 . 8 ± 0 . 4 -- sf120 ( pfb3 ) ompt degp ptr m9 6 . 4 ± 0 . 8 33__________________________________________________________________________ the effect of zinc chloride addition to protease - deficient cells grown in minimal medium w salts is shown in table iv . protein a - β - lactamase expression from sf110 harboring plasmid vector pfb3 was increased by 46 % when 0 . 1 mm zncl 2 was added to the medium . the effect on sf120 cells harboring pfb3 was an increase of 53 % when grown under the same conditions . table iv__________________________________________________________________________effect of zinc chloride addition to the growth medium of cells deficientin multipleproteases on the expression of protein a - β - lactamase from theplasmid vector pfb3 . relative penicillinasestrain strain growth specific activity ( plasmid ) characteristics medium (% ± sd ) __________________________________________________________________________sf110 ( pfb3 ) ompt degp ptr . sup .+ w 100sf110 ( pfb3 ) ompt degp ptr . sup .+ w + 0 . 1 mm zncl . sub . 2 146 ± 6sf120 ( pfb3 ) ompt degp ptr w 100sf120 ( pfb3 ) ompt degp ptr w + 0 . 1 mm zncl . sub . 2 153 ± 9__________________________________________________________________________ triply protease - deficient sf120 cells transformed with the plasmid expression vector pfb3 were grown in complex medium lb to which up to 1 mm zinc chloride was added . as shown in fig7 maximum expression of protein a - β - lactamase was observed when approximately 0 . 5 mm zn + 2 was added to the medium . the effect of ph on protein a - β - lactamase expression in cells transformed with plasmid vector pbf3 was determined . fermentations were carried out in bioflo iii fermenters with a working volume of 2 liters and followed for 24 hrs . the ph was maintained at a constant value by automatic addition of 1 m hcl or 1 m naoh to growth medium lb supplemented with 0 . 2 % glucose and 50 μg / ml ampicillin . fermenters were inoculated with 25 or 50 ml of transformed e . coli strain ks474 , deficient in degp , or sf110 , deficient in ompt and degp . fig8 shows that maximum expression of the fusion protein was obtained when the ph was maintained between 5 . 5 and 6 . 0 . the present example outlines the procedure contemplated by the applicants to be useful for the successful preparation of a mutant bacterium deficient in four proteases . construction of mutant organisms will involve the following steps : ( 1 ) cloning and amplification of a gene or part of a gene of a protease ; ( 2 ) mutagenesis of the isolated gene sequence to allow easy selection of the mutants ; ( 3 ) recombination of the mutated gene sequence into the chromosome ; and ( 4 ) transfer of the mutation into a strain deficient in three proteases . protease vi is isolated and purified from escherichia coli using well known techniques ( 25 - 27 ). the amino terminal sequence of the purified protein is determined by gas phase sequencing . degenerate oligonucleotide primers corresponding to the sequence of the first six or seven n - terminal amino acids are prepared by automated solid phase synthesis ( 19 ). the primers are designed so that a suitable restriction site is generated upon annealing to a plasmid library of e . coli genomic dna . amplification is carried out by polymerase chain reaction ( pcr ) using an additional primer which corresponds to a plasmid sequence the amplified dna is isolated and subcloned within a suitable vector containing appropriate restriction sites . the gene or part of the gene thus amplified is then used to screen an ordered plasmid e . coli library so that the gene is identified in the physical map of the genome , subsequently allowing the entire gene to be cloned . once the protease gene has been cloned on a plasmid , mutations are generated either in vitro as described in example 2 or in vivo by employing transposon mutagenesis with tn10 which is a transposon carrying tetracycline resistance . a mutation resulting in deletion of a substantial fraction of the gene &# 39 ; s dna and linked to the antibiotic resistance gene is then isolated . the mutation is then transferred into the triple mutant strain sf120 to generate a quadruple mutant strain exhibiting substantial reduction in four protease activities . strains in which the transposon has been excised are isolated by screening for cells that have lost the selectable marker . these cells will not exhibit tetracycline resistance but will be defective in protease vi as well as ompt , degp and protease iii . the references listed below are incorporated herein by reference to the extent they supplement , explain , provide a background for or teach methodology , techniques and / or compositions employed therein . 2 . huse , w . d ., lakshmi , s ., iverson , s . a ., kang , a . s ., alting - 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