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New approaches to increase the expression and stability of cloned foreign genes in Escherichia coli
Journal of Biotechnology, 13 (1990) 243-250
243
Elsevier BIOTEC 00434
New approaches to increase the expression and stability of cloned foreign genes in Escherichia coli T a m a s Lukacsovich 1, Gabriella B a l i k o 1, A n d r a s Orosz 1, Eva Balla 2 and Pal Venetianer 1 I Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, H-6701, Hungary and 2 Chemical Works of Gedeon Richter Ltd., PO Box 27, Budapest 10, H-1475, Hungary
(Accepted 10 July 1989)
Summary A family of expression plasmid vectors were constructed by fusing the strong P2 promoter of the r r n B gene of E s c h e r i c h i a coil (coding for ribosomal RNA) to the lac operator, thereby eliminating regulatory sequences from the r r n B gene and placing the expression under lac repressor control. This promoter proved to be stronger in vivo than the well-known consensus tac promoter, and its strength could be further increased by converting the sequence to consensus. The stability of the recombinant proteins could be increased by fusion to various lengths of the N-terminal end of /~-galactosidase, or by inserting a synthetic oligonucleotide, coding for heptathreonine. A new method was developed for the stabilization of recombinant plasmids without antibiotic selection, based on the presence of an essential gene on the plasmid and its absence from the chromosome. The application of this method is illustrated by the example of a plasmid expressing human proinsulin. P2 Promoter; r r n B gene; Replacement; Terminator; Consensus
Introduction E s c h e r i c h i a coil is still the most frequently used host for the recombinant D N A technology-based industrial production of proteins and peptides. Although a very
Correspondence to: T. Lukacsovich, Institute of Biochemistry, Biological Research Center, PO Box 521, Szeged H-6701, Hungary. Presented at the 2nd International Symposium on Overproduction of Microbial Products, held in (~eske Bud~jovice, Czechoslovakia, 3-9 July 1988.
0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
244 large number of vectors have been developed for these purposes (Denhardt and Colasanti, 1987), new variants are still needed, because each gene to be cloned and expressed, represents an individual case, posing different problems for the biotechnology industry. In constructing the vector plasmids described in this communication, we addressed three problems: (a) maximizing the transcription of the cloned gene in an easily regulatable way; (b) increasing the stability of the synthesized protein or peptide; (c) increasing the stability of the recombinant plasmid. As part of this work was published elsewhere (Lukacsovich et al., 1989a,b; Lukacsovich and Venetianer, 1989), the first two problems will only be briefly summarized. Materials and Methods
Cells and plasmids E. coli JM107 (Yanisch-Perron et al., 1985) and C600 (Appleyard, 1954) cells were used throughout this work. Most of the plasmid constructions were described in detail in previous communications from this laboratory (Boros et al., 1986; Lukacsovich et al., 1989a, b; Lukacsovich and Venetianer, 1989). Plasmid pKLA1 (Orosz et al., in preparation) is a pBR322-based terminator-probe vector that contains the double transcriptional terminator of the rrnB gene. Plasmid pER23ATI contains the sequence coding for human proinsulin inserted into the ClaI site of the expression vector pER23AT (Lukacsovich et al., 1989b). Plasmid pKT101 is a ColEI compatible plasmid carrying a kanamycin-resistance marker and the lacI q gene (a gift from A. Kiss). Growth and media Laboratory experiments were carried out in yeast-tryptone medium at 37 ° C. Large scale experiments were carried out in a 100 1 reactor with fed-batch technology in rich medium, at 3 7 ° C for 12 h in the presence of 0.5% lactose. The yield was 100 g 1-1 wet, or 30 g 1 ~ dry biomass. D N A methods All recombinant D N A experiments were done according to the manual of Maniatis et al. (1982). Protein electrophoresis SDS-PAGE was performed according to Laemmli (1970). A lpha-peptide assay Promoter activity was generally measured by estimating the amount of synthesized alpha-peptide according to Miller (1972). Results
(a) Maximizing transcription The seven genes coding for ribosomal RNA biosynthesis in E. coli are the most intensively transcribed regions of DNA in vivo. Each of the seven genes have two
245 promoters (P1 and P2) that are about equally strong in vitro, but in vivo they are subject to different, not entirely understood control mechanisms (Nomura et al., 1984). Our vectors are based on the P2 promoter of the rrnB gene. In addition to the sensu stricto promoter, they contain an AT-rich upstream activator region (this increases in vivo promoter activity at least tenfold, Lukacsovich et al., 1989a), but they lack the sequence immediately downstream of the promoter, because it has been shown earlier that this region is responsible for some down-regulation of rRNA transcription (Lukacsovich et al., 1987). Controlled transcription is ensured by a lac operator sequence appropriately located immediately downstream of the promoter. Transcription is terminated by the strong double terminators T 1 and T 2, also derived from the rrnB gene. Promoter activity is repressed by the presence of the lacI q gene either on the chromosome (JM107 host cell) or on a plasmid (pKT101). This can be induced in the laboratory by the gratuitous inducer I P T G by lactose. The in vivo rate of transcription in the fully induced state from this promoter is equal or higher than that from the well-known 'consensus' tac promoter (DeBoer et al., 1983). If however, the sequence of this P2 promoter is converted by site-directed mutagenesis to 'consensus', the rate of transcription is further increased 2-3-fold (Lukacsovich et al., 1989a).
(b) Maximizing protein stability It is well known that foreign proteins, especially smaller peptides, are often rapidly degraded intracellularly by proteolytic enzymes in E. coli. As a result of this process, the cloned genes are expressed, but the protein or peptide products do not accumulate in sufficient amounts for preparative purposes. This problem can be circumvented by fusing the cloned gene to a bacterial gene, or part of it. For this reason we constructed vectors coding for 66, 279, or 409 amino acid long bacterial proteins (mostly fl-galactosidase) as N-terminal fusion partners between the promoter and the foreign gene. When we tried these vectors with the human proinsulin gene, the 279 and 409 amino acid long fusion peptides proved to be sufficiently long to protect the proinsulin from degradation but the 66 amino acid long peptide was insufficient. Such long fusion partners are obviously undesirable, therefore we sought to increase stability with short N-terminal fusion partners. This goal was achieved by inserting a short synthetic heptathreonine-coding oligonucleotide between the first few codons of fl-galactosidase and the cloning site (Lukacsovich et al., 1989b). This vector, pER23AT, was used to express and accumulate human proinsulin. As Fig. 1 shows, the accumulation of the fusion protein (70% of which was human proinsulin) was high on the pilot-plant scale. The schematic structures of all the vectors described here are summarized in Fig. 2. (c) Increasing plasmid stability The high-level expression of a cloned foreign gene is usually harmful for the cell. Therefore recombinant plasmids are lost from the population at a rapid rate during continuous growth, unless a strong selection pressure is provided to preserve the
246
9
8
10
Fig. 1. Accumulation of the proinsulin fusion protein during growth. E. coli cells transformed with pER23ATI were grown in a 100 1 bioreactor as described in Materials and Methods. Samples were taken every hour and total cellular protein was analyzed on 20% SDS-polyacrylamide gels. Lanes: 1, molecular mass markers (94, 67, 43, 30, 20.1, 14.4 kDa); 2, sample before induction; 3-10, samples 1 - 8 h after induction.
•0
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Fig. 2. Schematic structure of the pER23 plasrnid and its derivatives. Open bars represent the elements of the expression cassette, inserted into the pBR322 plasmid (solid line) between positions 4279 and 2069. Functionally important regions are indicated by dots. The coding regions are indicated by braces, and the length of the translation products is given (aa: amino acid residue). T l, T 2, terminators; ori, origin of plasmid replication; Ap R, fl-lactamase gene; P2, promoter of the rrnB gene; ©, lac operator. The localization of restriction sites that can be used for cloning foreign genes is indicated: P, PuulI; H, HindlII; C, ClaI; E, EcoRI; B, BamHl; Ps, Pstl; Bg, BgllI; X, XbaI; Hp, Hpal.
247 -
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Fig. 3. Construction of the stabilized, pER23LCTI plasmid, expressing human proinsulin. Steps of the construction: (1) pER234C was constructed by isolating a 750 bp Taq! fragment from pBR329 (Covarrubbias and Bolivar, 1982), and inserting it into the Cla[ site of pER234 (Lukacsovich et al., 1989b); (2) pER23LICAT (Lukacsovich and Venetianer, 1989) was cleaved with EcoRI and tIindIII, and the smaller fragment was inserted into similarly digested pUC18 (Yanisch-PeFron et al., 1985); (3) the resulting pUC18L plasmid was partially digested with Pvull and a BamHI linker was inserted into the PvuII site upstream of the lacUV5 promoter; (4) this plasmid was cleaved with HindIII, and the smaller fragment obtained from the HindIII digestion of pER234C was inserted; (5) the resulting pUC18LC plasmid was partially digested with BglII, and a terminator fragment, obtained from pKLA1 (Orosz et al., in preparation) by BamHI digestion was inserted in the middle of the L1 gene; (6) the resulting pUC18LCT plasmid was digested with BamHI, and the smaller fragment was inserted into the BclI site of pER23ATI. Ap, ampicillin-resistance gene; Tc, tetracycline-resistance gene; CAT, chloramphenicol-resistance gene; a, alpha-peptide of fl-galactosidase; L1, Lll, genes coding for ribosomal proteins; P2, ribosomal RNA P2 promoter; T t, T 2, ribosomal RNA transcriptional terminators; UV5, UV5 promoter of the lac operon. Only those restriction enzyme cleavage sites are shown, which were relevant for the construction.
248 plasmids. This is usually d o n e b y using antibiotics in the m e d i u m , a n d the presence of resistance genes on the r e c o m b i n a n t plasmids. This procedure however increases the costs of p r o d u c t i o n . I n order to c i r c u m v e n t this problem, we recently developed a m e t h o d that can be generally used, w i t h o u t a p p l y i n g special host strains, a n d that selects for the presence of the p l a s m i d w i t h o u t antibiotics. T h e essence of the new m e t h o d is that: (1) a n essential bacterial gene is p u t o n the p l a s m i d ; (2) the f u n c t i o n of this gene is b l o c k e d by the insertion of a strong t r a n s c r i p t i o n a l t e r m i n a t o r ; (3) as a result of d o u b l e h o m o l o g o u s r e c o m b i n a t i o n , the i n c a p a c i t a t e d gene is exchanged with its f u n c t i o n a l c h r o m o s o m a l allele. T h u s the cell in which this exchange took place will n o t be viable w i t h o u t the plasmid, because a n essential gene will be present only there. The general m e t h o d was described elsewhere (Lukacsovich a n d Venetianer, 1989), here we describe its application for the p r o i n s u l i n - p r o d u c i n g plasmid.
123
4
5
6
Fig. 4. Effect of essential gene-replacement on plasmid stability. IPTG was added to cell cultures at an OD550 = 0.4. Cells were harvested 6 h later. Total protein extracts were run on 15% SDS-polyacrylamide gels according to Laemmli (1970). Lanes: 1, E. coli C600 (pKT101) without recombinant plasmid; 2, E. coli C600 (pKT101) transformed with pER23LCTI, grown in the presence of ampicillin, without induction; 3, E. coli C600 (pKT101) transformed with pER23LCTI, grown in the presence of ampicillin, after induction with 3 mM IPTG; 4, E. coil C600 (pKT101) transformed with pER23LCTl, grown in the absence of ampicillin, after induction with 3 mM IPTG; 5, E. coli C600 (pKT101) transformed with pER23LCTI and selected for recombinants on chloramphenicol plates, recombinants grown in the absence of ampicillin, after induction with 3 mM IPTG; 6, molecular mass markers (94, 67, 43, 30, 20.1, 14.4 kDa).
249 Plasmid pER23LCTI was constructed by the procedure illustrated and described in Fig. 3. It has the following properties. It contains the human proinsulin gene in fusion with a short semisynthetic region coding for the first few N-terminal amino acids of fl-galactosidase and seven threonines separated by a methionine from the proinsulin. This gene is under the control of the rrnB P2 promoter, the lac operator, the lac translation signals and the rrnB transcriptional terminators. It contains the ampicillin-resistance gene from pBR322, and an artificial operon under the control of the lacUV5 promoter. The operon contains the essential bacterial gene coding for the L1 ribosomal protein (and part of the L11 ribosomal protein gene), interrupted by a strong transcriptional terminator, derived also from the rrnB gene. The second gene of this operon originates from plasmid pBR329 (Covarrubbias and Bolivar, 1982), and codes for the chloramphenicol transacetylase enzyme. This gene is not expressed on the plasmid, because of the absolute polarity caused by the terminators. E. coli C600 cells (harboring plasmid pKT101, to ensure repression of the cloned gene) were transformed by pER23LCTI. The transformants were resistant to ampicillin and kanamycin, but sensitive to chloramphenicol. These cells were grown overnight in the presence of 100 /~g ml -a ampicillin and 50/~g ml -~ kanamycin. Then a 150 #l aliquot was spread on plates containing 200/~g ml-1 chloramphenicol. On these plates only such colonies could survive, in which the required double recombination event took place, i.e., the intact chromosomal L1 gene replaced the insertionally inactivated copy on the plasmid. Physical mapping of plasmids isolated from the resistant colonies verified this assumption. In these recombinants, the plasmids were stably maintained, without any antibiotics, even after induction of the proinsulin gene. On the other hand, prolonged growth under inducing conditions, without antibiotics, leads to the rapid loss of the original plasmids (i.e., those before the double recombination). This phenomenon is illustrated in Fig. 4.
Discussion
The vector constructs described here represent the results of an effort to optimize the production of recombinant human proinsulin. All constructs were, however, developed with a broader application in mind, namely to obtain a series of vectors that can be used for a wide range of recombinant genes. Although the principles applied were not entirely new (Brosius and Holy, 1984), we believe that in many ways this family of vehicles represents a useful new tool for the practicing biotechnologist. The advantages can be briefly summarized as follows. (1) The Pa promoter with its activating upstream region, especially if its sequence is converted to consensus, is certainly one of the strongest promoters in E. coli, giving an extremely high rate of in vivo expression. (2) It is well known that the efficiency of the lac operator-repressor to prevent transcription is very dependent on the exact position of the operator, and also depends on the sequence of the promoter. In our vectors the repression is efficient, giving approximately 1 : 300 repressed/induced ratio.
250 (3) T h e v a r y i n g l e n g t h o f a v a i l a b l e N - t e r m i n a l f u s i o n p a r t n e r s , e s p e c i a l l y t h e short N-terminal plus heptathreonine provided by pER23AT, provides possibilities to a c c u m u l a t e o t h e r w i s e u n s t a b l e s h o r t p e p t i d e s . (4) T h e e s s e n t i a l g e n e - r e p l a c e m e n t m e t h o d o f s t a b i l i z i n g p l a s m i d s saves p r o c e s s c o s t s a n d is a p p l i c a b l e f o r a n y h o s t strain. Some examples of the application of these vectors: HIV virus-processing protease ( G i a m a n d Boros, 1988), v a s o a c t i v e i n t e s t i n a l p o l y p e p t i d e ( S i m o n c s i t s et al., 1988), t u m o r n e c r o s i s f a c t o r ( M a i et al., Pers. C o m m u n . ) , s y n t h e t i c h u m a n i n s u l i n A a n d B c h a i n ( S i m o n c s i t s et al., Pers. C o m m u n . ) , c h i c k e n c a r t i l a g e m a t r i x p r o t e i n ( D e a k et al., Pers. C o m m u n . ) , firefly l u c i f e r a s e ( L u k a c s o v i c h , U n p u b l . ) a n d a l a r g e n u m b e r of c l o n e d b a c t e r i a l genes.
References Appleyard, R.K. (1954) Segregation of new lysogenic types during growth of a double lysogenic strain derived from Escherichia coli K12. Genetics 39, 440-452. Boros, I., Lukacsovich, T., Baliko, G. and Venetianer, P. (1986) Expression vectors based on the rac fusion promoter. Gene 42, 97-100. Brosius, J. and Holy, A. (1984) Regulation of ribosomal RNA promoters with a synthetic lac operator. Proc. Natl. Acad. Sci. USA 81, 6929-6933. Covarrubias, L. and Bolivar, F. (1982) Construction and characterization of new cloning vehicles. VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication. Gene 17, 79-89. DeBoer, H.A., Comstock, L.J. and Vasser, M. (•983) The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc. Natl. Acad. Sci. USA 80, 21-25. Denhardt, D.T. and Colasanti, J. (1987) A survey of vectors for regulating expression of cloned DNA in E. coll. In: Rodriguez, R.L. and Denhardt, D.T. (Eds.), Vectors, Butterworths, Stoneham, MA, pp. 179-204. Giam, C.-Z. and Boros, I. (1988) In vivo and in vitro autoprocessing of human immunodeficiency virus protease expressed in Escherichia coil J. Biol. Chem. 263, 14617-14620. Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685. Lukacsovich, T. and Venetianer, P. (1989) New method for the targeted inactivation of essential bacterial genes. Mol. Gen. Genet., in press. Lukacsovich, T., Boros, I. and Venetianer, P. (1987) New regulatory features of the promoters of an E. coli r R N A gene. J. Bacteriol. 169, 272-277. Lukacsovich, T., Gaal, T. and Venetianer, P. (1989a) The structural basis of the high in vivo strength of the rRNA P2 promoter of Escherichia coll. Gene, in press. Lukacsovich, T., Bahko, G., Orosz, A. and Venetianer, P. (1989b) A family of expression vectors based on the rrnB P2 promoter of Escherichia coli. Submitted for publication. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Nomura, M., Gourse, R. and Baughman, R. (1984) Regulation of the synthesis of ribosomes and ribosomal components. Annu. Rev. Biochem. 53, 75-117. Simoncsits, A., Tjrrnhammar, M.-L., Kalman, M., Cserpan, I., Gafvelin, G. and Bartfai, T. (1988) Synthesis, cloning and expression in Escherichia coli of artificial genes coding for biologically active elongated precursors of the vasoactive intestinal polypeptide. Eur. J. Biochem. 178, 343-350. Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mpl8 and pUC19 vectors. Gene 33, 103-119.
243
Elsevier BIOTEC 00434
New approaches to increase the expression and stability of cloned foreign genes in Escherichia coli T a m a s Lukacsovich 1, Gabriella B a l i k o 1, A n d r a s Orosz 1, Eva Balla 2 and Pal Venetianer 1 I Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, H-6701, Hungary and 2 Chemical Works of Gedeon Richter Ltd., PO Box 27, Budapest 10, H-1475, Hungary
(Accepted 10 July 1989)
Summary A family of expression plasmid vectors were constructed by fusing the strong P2 promoter of the r r n B gene of E s c h e r i c h i a coil (coding for ribosomal RNA) to the lac operator, thereby eliminating regulatory sequences from the r r n B gene and placing the expression under lac repressor control. This promoter proved to be stronger in vivo than the well-known consensus tac promoter, and its strength could be further increased by converting the sequence to consensus. The stability of the recombinant proteins could be increased by fusion to various lengths of the N-terminal end of /~-galactosidase, or by inserting a synthetic oligonucleotide, coding for heptathreonine. A new method was developed for the stabilization of recombinant plasmids without antibiotic selection, based on the presence of an essential gene on the plasmid and its absence from the chromosome. The application of this method is illustrated by the example of a plasmid expressing human proinsulin. P2 Promoter; r r n B gene; Replacement; Terminator; Consensus
Introduction E s c h e r i c h i a coil is still the most frequently used host for the recombinant D N A technology-based industrial production of proteins and peptides. Although a very
Correspondence to: T. Lukacsovich, Institute of Biochemistry, Biological Research Center, PO Box 521, Szeged H-6701, Hungary. Presented at the 2nd International Symposium on Overproduction of Microbial Products, held in (~eske Bud~jovice, Czechoslovakia, 3-9 July 1988.
0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
244 large number of vectors have been developed for these purposes (Denhardt and Colasanti, 1987), new variants are still needed, because each gene to be cloned and expressed, represents an individual case, posing different problems for the biotechnology industry. In constructing the vector plasmids described in this communication, we addressed three problems: (a) maximizing the transcription of the cloned gene in an easily regulatable way; (b) increasing the stability of the synthesized protein or peptide; (c) increasing the stability of the recombinant plasmid. As part of this work was published elsewhere (Lukacsovich et al., 1989a,b; Lukacsovich and Venetianer, 1989), the first two problems will only be briefly summarized. Materials and Methods
Cells and plasmids E. coli JM107 (Yanisch-Perron et al., 1985) and C600 (Appleyard, 1954) cells were used throughout this work. Most of the plasmid constructions were described in detail in previous communications from this laboratory (Boros et al., 1986; Lukacsovich et al., 1989a, b; Lukacsovich and Venetianer, 1989). Plasmid pKLA1 (Orosz et al., in preparation) is a pBR322-based terminator-probe vector that contains the double transcriptional terminator of the rrnB gene. Plasmid pER23ATI contains the sequence coding for human proinsulin inserted into the ClaI site of the expression vector pER23AT (Lukacsovich et al., 1989b). Plasmid pKT101 is a ColEI compatible plasmid carrying a kanamycin-resistance marker and the lacI q gene (a gift from A. Kiss). Growth and media Laboratory experiments were carried out in yeast-tryptone medium at 37 ° C. Large scale experiments were carried out in a 100 1 reactor with fed-batch technology in rich medium, at 3 7 ° C for 12 h in the presence of 0.5% lactose. The yield was 100 g 1-1 wet, or 30 g 1 ~ dry biomass. D N A methods All recombinant D N A experiments were done according to the manual of Maniatis et al. (1982). Protein electrophoresis SDS-PAGE was performed according to Laemmli (1970). A lpha-peptide assay Promoter activity was generally measured by estimating the amount of synthesized alpha-peptide according to Miller (1972). Results
(a) Maximizing transcription The seven genes coding for ribosomal RNA biosynthesis in E. coli are the most intensively transcribed regions of DNA in vivo. Each of the seven genes have two
245 promoters (P1 and P2) that are about equally strong in vitro, but in vivo they are subject to different, not entirely understood control mechanisms (Nomura et al., 1984). Our vectors are based on the P2 promoter of the rrnB gene. In addition to the sensu stricto promoter, they contain an AT-rich upstream activator region (this increases in vivo promoter activity at least tenfold, Lukacsovich et al., 1989a), but they lack the sequence immediately downstream of the promoter, because it has been shown earlier that this region is responsible for some down-regulation of rRNA transcription (Lukacsovich et al., 1987). Controlled transcription is ensured by a lac operator sequence appropriately located immediately downstream of the promoter. Transcription is terminated by the strong double terminators T 1 and T 2, also derived from the rrnB gene. Promoter activity is repressed by the presence of the lacI q gene either on the chromosome (JM107 host cell) or on a plasmid (pKT101). This can be induced in the laboratory by the gratuitous inducer I P T G by lactose. The in vivo rate of transcription in the fully induced state from this promoter is equal or higher than that from the well-known 'consensus' tac promoter (DeBoer et al., 1983). If however, the sequence of this P2 promoter is converted by site-directed mutagenesis to 'consensus', the rate of transcription is further increased 2-3-fold (Lukacsovich et al., 1989a).
(b) Maximizing protein stability It is well known that foreign proteins, especially smaller peptides, are often rapidly degraded intracellularly by proteolytic enzymes in E. coli. As a result of this process, the cloned genes are expressed, but the protein or peptide products do not accumulate in sufficient amounts for preparative purposes. This problem can be circumvented by fusing the cloned gene to a bacterial gene, or part of it. For this reason we constructed vectors coding for 66, 279, or 409 amino acid long bacterial proteins (mostly fl-galactosidase) as N-terminal fusion partners between the promoter and the foreign gene. When we tried these vectors with the human proinsulin gene, the 279 and 409 amino acid long fusion peptides proved to be sufficiently long to protect the proinsulin from degradation but the 66 amino acid long peptide was insufficient. Such long fusion partners are obviously undesirable, therefore we sought to increase stability with short N-terminal fusion partners. This goal was achieved by inserting a short synthetic heptathreonine-coding oligonucleotide between the first few codons of fl-galactosidase and the cloning site (Lukacsovich et al., 1989b). This vector, pER23AT, was used to express and accumulate human proinsulin. As Fig. 1 shows, the accumulation of the fusion protein (70% of which was human proinsulin) was high on the pilot-plant scale. The schematic structures of all the vectors described here are summarized in Fig. 2. (c) Increasing plasmid stability The high-level expression of a cloned foreign gene is usually harmful for the cell. Therefore recombinant plasmids are lost from the population at a rapid rate during continuous growth, unless a strong selection pressure is provided to preserve the
246
9
8
10
Fig. 1. Accumulation of the proinsulin fusion protein during growth. E. coli cells transformed with pER23ATI were grown in a 100 1 bioreactor as described in Materials and Methods. Samples were taken every hour and total cellular protein was analyzed on 20% SDS-polyacrylamide gels. Lanes: 1, molecular mass markers (94, 67, 43, 30, 20.1, 14.4 kDa); 2, sample before induction; 3-10, samples 1 - 8 h after induction.
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Fig. 2. Schematic structure of the pER23 plasrnid and its derivatives. Open bars represent the elements of the expression cassette, inserted into the pBR322 plasmid (solid line) between positions 4279 and 2069. Functionally important regions are indicated by dots. The coding regions are indicated by braces, and the length of the translation products is given (aa: amino acid residue). T l, T 2, terminators; ori, origin of plasmid replication; Ap R, fl-lactamase gene; P2, promoter of the rrnB gene; ©, lac operator. The localization of restriction sites that can be used for cloning foreign genes is indicated: P, PuulI; H, HindlII; C, ClaI; E, EcoRI; B, BamHl; Ps, Pstl; Bg, BgllI; X, XbaI; Hp, Hpal.
247 -
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Fig. 3. Construction of the stabilized, pER23LCTI plasmid, expressing human proinsulin. Steps of the construction: (1) pER234C was constructed by isolating a 750 bp Taq! fragment from pBR329 (Covarrubbias and Bolivar, 1982), and inserting it into the Cla[ site of pER234 (Lukacsovich et al., 1989b); (2) pER23LICAT (Lukacsovich and Venetianer, 1989) was cleaved with EcoRI and tIindIII, and the smaller fragment was inserted into similarly digested pUC18 (Yanisch-PeFron et al., 1985); (3) the resulting pUC18L plasmid was partially digested with Pvull and a BamHI linker was inserted into the PvuII site upstream of the lacUV5 promoter; (4) this plasmid was cleaved with HindIII, and the smaller fragment obtained from the HindIII digestion of pER234C was inserted; (5) the resulting pUC18LC plasmid was partially digested with BglII, and a terminator fragment, obtained from pKLA1 (Orosz et al., in preparation) by BamHI digestion was inserted in the middle of the L1 gene; (6) the resulting pUC18LCT plasmid was digested with BamHI, and the smaller fragment was inserted into the BclI site of pER23ATI. Ap, ampicillin-resistance gene; Tc, tetracycline-resistance gene; CAT, chloramphenicol-resistance gene; a, alpha-peptide of fl-galactosidase; L1, Lll, genes coding for ribosomal proteins; P2, ribosomal RNA P2 promoter; T t, T 2, ribosomal RNA transcriptional terminators; UV5, UV5 promoter of the lac operon. Only those restriction enzyme cleavage sites are shown, which were relevant for the construction.
248 plasmids. This is usually d o n e b y using antibiotics in the m e d i u m , a n d the presence of resistance genes on the r e c o m b i n a n t plasmids. This procedure however increases the costs of p r o d u c t i o n . I n order to c i r c u m v e n t this problem, we recently developed a m e t h o d that can be generally used, w i t h o u t a p p l y i n g special host strains, a n d that selects for the presence of the p l a s m i d w i t h o u t antibiotics. T h e essence of the new m e t h o d is that: (1) a n essential bacterial gene is p u t o n the p l a s m i d ; (2) the f u n c t i o n of this gene is b l o c k e d by the insertion of a strong t r a n s c r i p t i o n a l t e r m i n a t o r ; (3) as a result of d o u b l e h o m o l o g o u s r e c o m b i n a t i o n , the i n c a p a c i t a t e d gene is exchanged with its f u n c t i o n a l c h r o m o s o m a l allele. T h u s the cell in which this exchange took place will n o t be viable w i t h o u t the plasmid, because a n essential gene will be present only there. The general m e t h o d was described elsewhere (Lukacsovich a n d Venetianer, 1989), here we describe its application for the p r o i n s u l i n - p r o d u c i n g plasmid.
123
4
5
6
Fig. 4. Effect of essential gene-replacement on plasmid stability. IPTG was added to cell cultures at an OD550 = 0.4. Cells were harvested 6 h later. Total protein extracts were run on 15% SDS-polyacrylamide gels according to Laemmli (1970). Lanes: 1, E. coli C600 (pKT101) without recombinant plasmid; 2, E. coli C600 (pKT101) transformed with pER23LCTI, grown in the presence of ampicillin, without induction; 3, E. coli C600 (pKT101) transformed with pER23LCTI, grown in the presence of ampicillin, after induction with 3 mM IPTG; 4, E. coil C600 (pKT101) transformed with pER23LCTl, grown in the absence of ampicillin, after induction with 3 mM IPTG; 5, E. coli C600 (pKT101) transformed with pER23LCTI and selected for recombinants on chloramphenicol plates, recombinants grown in the absence of ampicillin, after induction with 3 mM IPTG; 6, molecular mass markers (94, 67, 43, 30, 20.1, 14.4 kDa).
249 Plasmid pER23LCTI was constructed by the procedure illustrated and described in Fig. 3. It has the following properties. It contains the human proinsulin gene in fusion with a short semisynthetic region coding for the first few N-terminal amino acids of fl-galactosidase and seven threonines separated by a methionine from the proinsulin. This gene is under the control of the rrnB P2 promoter, the lac operator, the lac translation signals and the rrnB transcriptional terminators. It contains the ampicillin-resistance gene from pBR322, and an artificial operon under the control of the lacUV5 promoter. The operon contains the essential bacterial gene coding for the L1 ribosomal protein (and part of the L11 ribosomal protein gene), interrupted by a strong transcriptional terminator, derived also from the rrnB gene. The second gene of this operon originates from plasmid pBR329 (Covarrubbias and Bolivar, 1982), and codes for the chloramphenicol transacetylase enzyme. This gene is not expressed on the plasmid, because of the absolute polarity caused by the terminators. E. coli C600 cells (harboring plasmid pKT101, to ensure repression of the cloned gene) were transformed by pER23LCTI. The transformants were resistant to ampicillin and kanamycin, but sensitive to chloramphenicol. These cells were grown overnight in the presence of 100 /~g ml -a ampicillin and 50/~g ml -~ kanamycin. Then a 150 #l aliquot was spread on plates containing 200/~g ml-1 chloramphenicol. On these plates only such colonies could survive, in which the required double recombination event took place, i.e., the intact chromosomal L1 gene replaced the insertionally inactivated copy on the plasmid. Physical mapping of plasmids isolated from the resistant colonies verified this assumption. In these recombinants, the plasmids were stably maintained, without any antibiotics, even after induction of the proinsulin gene. On the other hand, prolonged growth under inducing conditions, without antibiotics, leads to the rapid loss of the original plasmids (i.e., those before the double recombination). This phenomenon is illustrated in Fig. 4.
Discussion
The vector constructs described here represent the results of an effort to optimize the production of recombinant human proinsulin. All constructs were, however, developed with a broader application in mind, namely to obtain a series of vectors that can be used for a wide range of recombinant genes. Although the principles applied were not entirely new (Brosius and Holy, 1984), we believe that in many ways this family of vehicles represents a useful new tool for the practicing biotechnologist. The advantages can be briefly summarized as follows. (1) The Pa promoter with its activating upstream region, especially if its sequence is converted to consensus, is certainly one of the strongest promoters in E. coli, giving an extremely high rate of in vivo expression. (2) It is well known that the efficiency of the lac operator-repressor to prevent transcription is very dependent on the exact position of the operator, and also depends on the sequence of the promoter. In our vectors the repression is efficient, giving approximately 1 : 300 repressed/induced ratio.
250 (3) T h e v a r y i n g l e n g t h o f a v a i l a b l e N - t e r m i n a l f u s i o n p a r t n e r s , e s p e c i a l l y t h e short N-terminal plus heptathreonine provided by pER23AT, provides possibilities to a c c u m u l a t e o t h e r w i s e u n s t a b l e s h o r t p e p t i d e s . (4) T h e e s s e n t i a l g e n e - r e p l a c e m e n t m e t h o d o f s t a b i l i z i n g p l a s m i d s saves p r o c e s s c o s t s a n d is a p p l i c a b l e f o r a n y h o s t strain. Some examples of the application of these vectors: HIV virus-processing protease ( G i a m a n d Boros, 1988), v a s o a c t i v e i n t e s t i n a l p o l y p e p t i d e ( S i m o n c s i t s et al., 1988), t u m o r n e c r o s i s f a c t o r ( M a i et al., Pers. C o m m u n . ) , s y n t h e t i c h u m a n i n s u l i n A a n d B c h a i n ( S i m o n c s i t s et al., Pers. C o m m u n . ) , c h i c k e n c a r t i l a g e m a t r i x p r o t e i n ( D e a k et al., Pers. C o m m u n . ) , firefly l u c i f e r a s e ( L u k a c s o v i c h , U n p u b l . ) a n d a l a r g e n u m b e r of c l o n e d b a c t e r i a l genes.
References Appleyard, R.K. (1954) Segregation of new lysogenic types during growth of a double lysogenic strain derived from Escherichia coli K12. Genetics 39, 440-452. Boros, I., Lukacsovich, T., Baliko, G. and Venetianer, P. (1986) Expression vectors based on the rac fusion promoter. Gene 42, 97-100. Brosius, J. and Holy, A. (1984) Regulation of ribosomal RNA promoters with a synthetic lac operator. Proc. Natl. Acad. Sci. USA 81, 6929-6933. Covarrubias, L. and Bolivar, F. (1982) Construction and characterization of new cloning vehicles. VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication. Gene 17, 79-89. DeBoer, H.A., Comstock, L.J. and Vasser, M. (•983) The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc. Natl. Acad. Sci. USA 80, 21-25. Denhardt, D.T. and Colasanti, J. (1987) A survey of vectors for regulating expression of cloned DNA in E. coll. In: Rodriguez, R.L. and Denhardt, D.T. (Eds.), Vectors, Butterworths, Stoneham, MA, pp. 179-204. Giam, C.-Z. and Boros, I. (1988) In vivo and in vitro autoprocessing of human immunodeficiency virus protease expressed in Escherichia coil J. Biol. Chem. 263, 14617-14620. Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685. Lukacsovich, T. and Venetianer, P. (1989) New method for the targeted inactivation of essential bacterial genes. Mol. Gen. Genet., in press. Lukacsovich, T., Boros, I. and Venetianer, P. (1987) New regulatory features of the promoters of an E. coli r R N A gene. J. Bacteriol. 169, 272-277. Lukacsovich, T., Gaal, T. and Venetianer, P. (1989a) The structural basis of the high in vivo strength of the rRNA P2 promoter of Escherichia coll. Gene, in press. Lukacsovich, T., Bahko, G., Orosz, A. and Venetianer, P. (1989b) A family of expression vectors based on the rrnB P2 promoter of Escherichia coli. Submitted for publication. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Nomura, M., Gourse, R. and Baughman, R. (1984) Regulation of the synthesis of ribosomes and ribosomal components. Annu. Rev. Biochem. 53, 75-117. Simoncsits, A., Tjrrnhammar, M.-L., Kalman, M., Cserpan, I., Gafvelin, G. and Bartfai, T. (1988) Synthesis, cloning and expression in Escherichia coli of artificial genes coding for biologically active elongated precursors of the vasoactive intestinal polypeptide. Eur. J. Biochem. 178, 343-350. Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mpl8 and pUC19 vectors. Gene 33, 103-119.