Patent Application: US-40365199-A

Abstract:
the invention relates to a regulation system for inducible expression of genes , comprising a lambdoid promoter , a gene coding for a repressor for the lambdoid promoter and a gene coding for an antirepressor of the repressor , which antirepressor is under the influence of an inducible promoter . the invention further relates to a regulatory replicon , comprising said gene coding for an antirepressor , an expression system , comprising said regulatory replicon , and an expression vector based on a lambdoid promoter , and also to a method for producing a gene product in a heterologous host , by providing a culture of a host comprising a heterologous sequence which codes for the gene product . providing a culture of a host comprising a heterologous sequence is obtained by putting the expression of the heterologous sequence under the control of a regulation system , a gene coding for a repressor for the lambdoid promoter and a gene coding for an antirepressor , and by inducing the promoter of the antirepressor gene .

Description:
following below is a summary of the definitions used in this application . antirepressor : protein which can neutralize a repressor and thus activates the repressed promoter . phage λ : ( bacteriophage lambda ) temperate bacteriophage which infects escherichia coli , belongs to the styloviridae family . phage p22 : ( bacteriophage p22 ) temperate phage which infects salmonella typhimurium , belongs to the podoviridae family . gene expression : expression of a gene by transcription and translation to a polypeptide or a protein ( functional protein ). temperate phage : phage which can pass on its genetic information through infection as well as via the cell division of the host after insertion in the genome . immunity : ( phage immunity ) superinfection resistance for phages with the same or homologous regulatory elements . lmbp : laboratory of molecular biology plasmid collection , recognized deposit body for plasmids , part of the belgian coordinated collection of micro - organisms ( bccm ). repressor : protein which prevents transcription initiation on one or more determined promoters . vector : a biological entity which can ensure the multiplication of genetic information . in the examples below the invention is illustrated on the basis of the prokaryote lacz gene and the eukaryote genes coding for human interferon - γ ( hifn γ ), murine interleukin 2 ( mil2 ) and human interleukin 2 ( hil2 ) as model system for protein synthesis . it will be apparent to a person skilled in the art that in an analogous manner other genes can be expressed in a regulated manner with the system described here without any inventive work having to be performed for this purpose . the materials and methods used are first elucidated hereinbelow . thereafter the invention is illustrated in the examples . in support of most of the methods reference is further made to sambrook et al . ( 1989 ) molecular cloning : a laboratory manual , 2nd ed . cold spring harbor laboratory press , cold spring harbor , usa , and miller , j . ( 1972 ) experiments in molecular genetics , cold spring harbor laboratory , new york . all cloning experiments were carried out in e . coli mc1061 ( hsdr mcrb arad139 δ ( araabc - leu ) 7697 δlacx74 galu galk rpsl thi ) ( casadaban and cohen , 1980 ) lysogenized with λ when multiplying λp l - containing plasmids . expression experiments were carried out in mci061 transformed with a regulatory plasmid ( for instance pci857 or pica2 ). the repressor came from pci857 ( lmbp537 ), a vector with a high copy number ( p15a replicon ), which carries an autogenously regulated ci857 gene coding for a thermosensitive ci mutant ( remaut et al ., 1983 ). s . typhimurium lt2 ( atcc 19 585 ) was grown in nutrient broth ( difco 0001 ) supplemented with 0 . 5 % nacl . phage p22 ( atcc 19 585 - b1 ) stocks were obtained from confluent lysis plates prepared by using an excess of plaque - forming units with freshly prepared nutrient agar plates . the macerated soft agar was cleared by centrifugation to isolate the phage particles . p22 dna was prepared by phenol extraction of purified phage particles . plr10t is a vector derived from plt10t ( mertens et al ., 1995 b ) in which the actual translation initiation site is preceded by a small , well - translated cistron which resulted from a fusion of the n - terminal region of t7g10 and the c - terminal piece of the e . coli trpb gene ( fig1 ). pdmi , 1 ( lmep1594 ) was obtained from dr . dietrich stüber ( hoffman - la roche , basel , switzerland ). ( see fig2 ) plasmid dna purification was carried out as described in sambrook et al ., 1989 . all enzymes used for plasmid cloning were obtained from new england biolabs or boehringer mannheim and used according to the recommendations of the supplier . the p22 ant region was amplified by means of pcr with vent dna polymerase ( new england biolabs ) using 5 ′- atcagaattcgcggtaacagtcagggcttcgg - 3 ′ ( seq . id no : 1 ) and forward and 5 ′- ttaaggatccgaagctgggtcgttgcgttgg - 3 ′ ( seq . id no : 2 ) as backward primer . this amplifies a 1054 bp dna region spanning the coordinates 498 - 1531 of the pop22imm genbank sequence ( sauer et al ., 1983 ). this includes the ant coding region with its own ribosomal binding site and adds a bamhi restriction site to the 3 ′ end . the amplified fragment was trimmed with bamhi and ligated in a pds12 vector opened with smai and bamhi ( stüber et al ., 1984 ). an xhoi - bamhi fragment from pds12ant ( with the pn 25 / o2 - ant combination ) was combined with the ci857 gene on an ecori - bamhi fragment derived from pat153ci857 ( mertens et al ., 1995 a ), ( lmbp1065 ), a laci q - containing fragment from puc181aci q ( lmbp3259 ) trimmed with aatii and ecori , and an aatii - sali pbr322 ( lmbp140 ) vector part . the resulting pical is a multi - copy number plasmid which contains a combination of laci q , ci857 and p n25 / o2 ant which is useful according to the invention . from this plasmid an eagi - psti fragment was combined with a puc18kan ( pharmacia , sweden ) trimmed with psti and xhoi and containing an npt gene ( km r ( kanamycin resistance )) and an xhoi - eagi vector fragment from plg339 ( stoker et al ., 1982 ) in order to obtain the plasmid pica2 according to the invention . pica2 ( fig1 ) is a low copy number plasmid compatible with all current expression vectors in respect of replication origin ( psc101 ) and antibiotic selection . expression strains containing pci857 , pica1 or pica2 were kept at a non - permissive temperature ( 28 ° c .). during manipulations preceding induction . λp l - dependent temperature induction was carried out in mc1061 with either pica2 according to the invention or the known pci857 by raising the culture temperature from 28 ° c . to 42 ° c . mc1061 ( pica1 ) and mc1061 ( pica2 ) were induced at temperatures of 28 ° c . or lower by adding iptg to a final concentration of 1 mm . the cells were harvested , resuspended in sonication buffer ( sb , 10 mm tris - cl ph 7 . 5 ; 0 . 1 m nacl ; 5 mm dtt ; 10 % glycerol ) and frozen at − 20 ° c . aliquots ( usually 200 μl ) were thawed at 37 ° c . and cooled on ice . the cells were subsequently opened by sonication on ice using a sonics & amp ; materials ( danbury conn ., usa ) sonicator with a microtip . lysates were then cleared by centrifugation at 15 000 g for 15 minutes . prior to cytokine assay the lysates were diluted in sb and filtered over a cellulose - acetate 0 . 22 μm pore - size filter . mil2 titers were determined by a proliferation assay using the il2 - dependent cytotoxic t - cell line ctll - 2 ( guisez et al ., 1993 ). human interferon - γ activity was determined on human fs4 cells by a cytopathic effect reduction assay using encephalomyocarditis virus as challenge virus ( devos et al ., 1982 ). the ant gene of phage p22 of s . typhimurium was amplified from purified p22 dna and cloned in the pds12 expression vector , as described in materials and methods . in this manner the gene was under the control of the p n25 / o2 promoter ( strueber et al ., 1984 ) inducible by means of iptg . induction of the resulting expression plasmid pds12 ant resulted in a high production of the repressor p22 ant ( fig4 b ). the ant gene was subsequently combined with laci q and λci857 in a manner such that the different promoters did not interfere with the expression of each individual gene . the resulting combination ( pica1 ) was transferred to a low copy number replicon which is cole1 - compatible and derived from psc101 and also carried a kanamycin resistance selection marker ( stoker et al ., 1982 ). the resulting plasmid pica2 contains all necessary information for repression of λp l or λp r ( by means of ci857 ) and repression ( by means of laci q ) and induction ( from the p n25 / o2 - ant promoter ) of the antirepressor , and can therefore be used as a suitable expression regulatory plasmid for iptg - induced λp l or λp r expression when it is combined with an expression plasmid containing a gene under the transcriptional control of the λp l or λp r promoter . tight regulation and expression at low temperatures with the p22 ant - based expression system in order to quantify the characteristics of the new expression system an expression vector was used containing as model gene a λp l - driven lacz gene for protein synthesis . this vector is capable of inducing high levels of functional b - galactosidase by means of translationally - coupled translation initiation ( mertens et al ., 1995 a ; mertens et al ., 1997 ). the non - induced levels of b - galactosidase , which could be influenced by a possible continuous presence of low non - induced levels of antirepressor protein , were comparably low when pica2 or pci857 ( without antirepressor ) plasmid were used . this means therefore that low , non - induced levels of antirepressor protein are probably not present , since if this were indeed the case a higher expression of lacz would be expected . investigation of the induction kinetics of the lacz gene was carried out in mc1061 [ pica2 ] at 18 ° c ., 24 ° c ., 28 ° c ., 37 ° c . or 42 ° c . after adding 1 mm iptg ( fig4 a ). temperatures above 28 ° c . likewise denature the temperature - sensitive ci857 λ - repressor and are an indication of the level of expression obtainable with this vector by thermo - induction . from fig4 a can be concluded that maximum b - galactosidase levels can be obtained by adding iptg at lower temperatures . as expected , the induction kinetics were slower at temperatures under 28 ° c ., because under these sub - optimal growth conditions a lower growth rate and a lower metabolism are obtained . fig4 b compares the level of the induced proteins in mc1061 [ pica2 ] [ plr10βgal ] and mc1061 [ pdmi , 1 ][ pds12 ant ], which shows that the high expression level of p22 ant obtained using the high copy number expression vector pds12 ant disappears by transferring the p n25 / o2 - ant combination to a low copy number plasmid ( 6 - 10 copies / cell ), while still retaining the ability to induce the lambdoid promoter by induction of ant . decreasing the synthesis of the repressor antagonist to & lt ; 1 % of the total protein synthesis is advantageous because the ultimate concern is to obtain a large quantity of a particular protein — the gene of which has been inserted behind the strong lambdoid promoter — and the ultimate yield of this target protein can be adversely affected if the repressor antagonist must be produced in large quantities . induction at low temperature can improve the production of soluble heterologous proteins induction resulting in a high expression level , particularly at increased temperatures where the e . coli metabolism is high , often results , as already indicated above , in the production of inclusion bodies , while at the same time further cell growth is often inhibited . for reasons which are still not entirely clear , induction at low temperatures is more favourable when production of correctly folded , soluble protein is required . ( lin et al ., 1990 ; shirano & amp ; shibata , 1990 ; schein and noteborn , 1988 ; bishia et al ., 1987 ; mizukami et al ., 1986 ). this example therefore investigates whether the different methods of induction ( temperature increase or iptg ) result in different quantities of soluble protein . used for this purpose were expression vectors derived from plt10t ( mertens et al ., 1995 ) containing one of the following genes : prokaryotic t7g10 or thioredoxin , two proteins which can be readily expressed in e . coli ); human interferon - γ ( hifnγ ) and murine interleukin - 2 ( mil2 ). fig5 compares production levels obtained either after thermo - induction or after low temperature iptg induction . it can be clearly inferred from the prokaryotic examples , t7g10 and thioredoxin , that in the same time - span the same quantity of heterologous protein can be induced . the strains induced by means of iptg continued to grow and eventually synthesized a larger quantity of host proteins ( fig5 b , lane b ). this resulted in a lower yield in comparison with the total protein content (% of the total protein ), but gave practically the same absolute yield ( mg protein per liter culture ). in the figure equivalent quantities of bacterial cultures are compared to each other . fig6 shows the activities of mil2 and hifnγ obtained after induction by temperature increase or by iptg induction . in these experiments with the eukaryotic proteins human interferon - γ and murine interleukin - 2 , two proteins which do aggregate readily in e . coli , less heterologous protein is formed after the iptg induction at low temperature ( fig5 a ). however , the high expression at 42 ° c . results exclusively in the forming of inclusion bodies , while after iptg induction at 28 ° c . a significant quantity of soluble protein is produced ( fig6 a and 6 b ). the soluble protein fraction visible on gel corresponds with the obtained amount of activity after a biological titration . in a preferred embodiment of the invention the synthesis of the repressor , the antirepressor and the repressor of the promoter controlling the gene of the antirepressor takes place from a low copy number plasmid . this example illustrates the use of the ant system from other replicons . as the case arises , the regulatory genes ( repressor ci857 controlled by the auto - regulatory promoter p m , antirepressor ant controlled by the p n25 / o2 promoter and the laci gene controlled by the constitutive p laci q - promoter ) were induced from a high copy number plasmid and a cole1 / pmb1 replication origin ( fig7 a ). the gene for expressing ( human interleukin 2 , hil2 ) was linked on a plasmid with a high copy number and a broad host range to the λp l promoter and a prokaryote ribosome binding site ( originating from the ner gene of phage mu ). this plasmid also contains an extra copy of the λp l repressor gene ci857 . the pplgnihil2 plasmid also contains the required functions also enabling replication in other bacteria ( fig7 b ). induction was investigated after adding 0 , 0 . 1 or 1 mm of the inducer iptg , which provides induction of ant from the p n25 / o2 promoter , which in turn brings about inhibition of the λp l , repressor ci857 and thus activates the λp l which results in overexpression of , in this case , hil2 ( fig7 c ). these inductions were carried out at 28 ° c . and compared with the classic temperature deactivation of ci857 by growing the bacteria further at 42 ° c . in this example the relative production of hil2 using the ant system and by temperature induction are roughly equal . the temperature induction at 42 ° c . was however found to cause a greater growth - inhibiting effect ( the gel shows equivalent samples of culture medium ). an ant - based expression system for the lambdoid promoters p22p l and p22p r induction of a repressor antagonist in order to induce a promoter can in principle also be applied to promoters other than the λp l . in this example a vector system is described which makes use of the p22 ant gene to deactivate the homologous p22c2 repressor and thus obtain induction of the p22p l or the p22p r . constructed first for this purpose was the pica3 plasmid which is a derivative of pica2 but which contains the p22c2 repressor gene instead of the λci857 repressor gene . the λp l promoter in plt10t3 was further replaced by both the p22p l ( plt22t3 ) and the p22p r ( prt22t3 ). both pica3 and plt22t3 and prt22t3 are shown in fig8 . an ant - based regulatory system for λp l and λp r that is inducible with l - arabinose in the pica2 regulatory plasmid , the induction of ant is controlled by the iptg - inducible p t5 n25 / o2 . control of ant - gene expression can in principle come from any inducible promoter . it is preferred that the promoter of choice is inducible also at lower temperatures ( 28 ° c . or lower ). it is also preferred that the promoter of choice be well regulated . if this would not be the case , the level of expression of the uninduced culture can be sufficient to initiate continuous expression . uncontrolled expression is likely to result in eventual loss of the functional expression strain . in this example a regulatory plasmid was constructed containing the promoter region of the e . coli arabinose operon ( p arabad ) and the gene encoding the arac repressor of this promoter . the ant - gene is in this way controlled by a promoter which is inducible by the addition of l - arabinose . the parabad promoter and the arac repressor gene encoding the repressor for this promoter were amplified from a wild type e . coli k12 bacterial strain , using the pcr primers nm73 ( atatatccaaggttatgcaatcgccatcgtttcactcc ) ( seq . id no : 3 ) and nm72 atatcggccgttatgacaacttgacggctacatc ( seq . id no : 4 ). pcr amplification was performed with vent dna polymerase ( new england biolabs ) and the resulting fragment was cloned between the xmaiii and the styl restriction sites present in pica2 . the resulting pica5 plasmid was characterized by restriction site mapping , pcr analysis and the pcr amplified insert was sequenced . fig9 shows the pica5 plasmid . comparison of the λp l - ant induction system with other iptg inducible expression systems although the λp l promoter is amongst the strongest promoters recognized by the e . coli transcriptional machinery , the t7 promoter , which is recognized by the extremely active t7 rna - polymerase ( t7rnap ), allows for a far greater amount of mrna to be formed ( studier and moffatt , 1986 ). a well - controlled , iptg - based induction system for the t7rnap that also resides on a psc101 - derived low - copy number plasmid system was previously described ( mertens et al ., 1995b ). the plt10mil2t and the plt10hifnγt expression plasmids were used , which contain both the λpl and the p t7 promoters ( mertens et al ., 1995a ), to compare the expression obtained from either the p t7 and the λp l induction system upon addition of iptg ( fig1 a ). strikingly , cells containing the t7 - based induction system stopped growing almost immediately after induction , while those induced by the λp l - ant system continued to proliferate . this resulted in an almost 10 - fold higher biomass after 4 h of induction at 28 ° c . remarkably , after induction of the p t7 t7rnap system all of the mil2 and almost all of the hifnγ were in the insoluble phase , while when using the λp l - ant system about 50 % of the heterologous protein was found in the soluble phase . the coding sequence of hifnγ combined with the strong rbst7g10 resulted in a favorable translation initiation region ( mertens et al ., 1995a ). when combined with a strong promoter on a high - copy number plasmid , abundant expression was obtained after induction . to emphasize the difference in promoter strength between various iptg - inducible promoters such as p trc ( amann et al ., 1988 ), p t5 n25 / o2 ( stüber et al ., 1984 ) and p t7 ( mertens et al ., 1995b ; studier et al ., 1990 ) and the λp l - ant system , the rbs - gene - terminator combination combined with the aforementioned promoters was transferred to an rk2 replicon ( blatny et al ., 1997 ). this resulted in expression plasmids with a much lower copy - number than the normally used cole1 - derived vectors . subsequently the induction of hifnγ was then compared using these vectors . using the stronger p t7 resulted in a higher level of production , but all of the hifnγ produced was insoluble . employing the p trc and p t5 n25 / o2 promoters did not result in visually detectable levels of induced protein from this low - copy number vector after sds - page staining with coomassie brilliant blue . however , when the λp l - ant system was used a clearly detectable level of huifnγ was synthesized , while the protein remained completely soluble ( fig1 b ). fig1 shows that induction at 28 ° c . using the λp l / ant system is more efficient in producing functional protein than other iptg - inducible expression systems . fig1 a demonstrates the sds - page analysis of soluble ( s ) and pelleted ( p ) proteins after induction of plt10mil2t or plt10hifnγt in either mc1061 [ pt7pol26 ] ( inducing the t7 promoter ) or mc1061 [ pica2 ] ( inducing the λp l - promoter ). induction was obtained by growing for 5 h at 28 ° c . in the presence of iptg . clearly , more soluble mil2 was obtained by using the λp l / ant induction system . unlike the t7 - system , the latter system also allowed the cultures to continue growing , and thus resulted in a higher biomass accumulation . fig1 b is a comparison of expression of the rbs t7g10 - hfnγ - t7tφ module combined with some different iptg - inducible promoters on an rk2 - derived low - copy number plasmid . 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