Tryptophan Promoter Derivatives on Multicopy Plasmids: A Comparative Analysis of Expression Potentials in Escherichia coli
MARTINE
LATTA, MICH\l=E`\LE PHILIT, ISABELLE MAURY, FABIENNE SOUBRIER, PATRICE DEN\l=E`\FLE, and JEAN-FRAN\l=C;\OIS MAYAUX
ABSTRACT A collection of variant plasmids expressing either Escherichia coli galactokinase or human serum albumin under the control of several E. coli trp promoter derivatives were constructed and studied for both efficiency of expression and regulation by tryptophan. Several variables, including the length of the upstream region, tandem duplications of a core promoter, and the insertion of the trp repressor trpR gene onto the expression vector, were studied. It is shown that derivatives containing sequences upstream from the \p=m-\35 region or multiple copies of the trp promoter produce twofold higher levels of protein than plasmids with a minimal trp promoter truncated at \p=m-\40. We show that the expression of a heterologous protein such as albumin can be significantly improved (13% vs. 7% of total proteins) if both the upstream trp promoter region, which enhances promoter strength, and an intact trpR gene, are included on the plasmids.
INTRODUCTION
(Landick and Yanofsky, 1987) and containing only the oppromoter elements (Harley and Reynolds, 1987). Although this 40-bp fragment contains all the sequence elements determining both the efficiency and regulation of transcriptional initiation (reviewed in Yanofsky et al, 1981), recent evidence indicates that elements outside the consensus core region might also be important for maximal transcription efficiency. For example, changing the first 20 nucleotides of the transcribed region may alter promoter strength 10-fold by influencing late events in transcriptional initiation (Kammerer et al, 1986). Comparison of strong E. coli promoters such as X PL, the lipoprotein (Ipp) or the Ptrp promoters indicated the presence of conserved A + T-rich blocks in the 5' upstream region (Nakamura and Inouye, 1979; Horn and Wells, 1981a); it has been proposed that these A + T-rich regions behave as constitutive transcriptional enhancers (Horn and Wells, 1981b; Nishi and Itoh, 1986). Poly(dA:dT) sequences have erator and core
(Ptrp) ÍS of the strongest regulated Escherichia coli promoters (Hallewell and Emtage, 1980; Russell and Bennett, 1982). It has often been used for the efficient expression of foreign proteins in E. coli (Nichols and Yanofsky, 1983; Tacon et al., 1983; Rosenberg et al, 1984). Recently, we reported that optimized expression vectors containing this promoter can be used in suitable E. coli strains to obtain high levels of intracytoplasmic expression of human proteins such as human serum albumin or tissue plasminogen activator (Latta et al, 1987; Sarmientos et al, 1989). Production of mature human interleukin 1/3 to several grams per liter of broth can be reached in high-cell density cultures of the E. coli B strain harboring such Ptrp expression plasmids (Jung et al, 1988). In particular, the nutritional requirements of wild-type strains such as E. coli B appeared more compatible with high-scale fermentation conditions at high biomass than those of current K12 laboratory strains. However, in these studies, we used a minimal Ptrp cassette (-39 to +1) lacking the attenuation control region
THE
PROMOTER OF THE TRYPTOPHAN OPERON
one
Laboratoire de
Génétique,
Institut de
also been shown to act as constitutive upstream promoter elements in yeast (reviewed in Struhl, 1986). To determine if transcriptional initiation from a tryptophan-regulated promoter can be improved further and to obtain higher production levels of heterologous proteins in
Biotechnologie, Rhône-Poulenc Santé,
94403
Vitry-sur-Seine Cedex, France.
E. coli from such promoters, we constructed several Ptrp derivatives containing either upstream regions of various lengths or multiple core promoter cassettes in tandem. We also studied the effect of the insertion of the trpR gene, coding for the Trp repressor, into the expression vectors. The in vivo strength and regulation of the different constructions were monitored both on the easily assayable E. coli galK gene and on the expression of heterologous HSA. Based on these results, we describe a new Ptrp vector that results in a stable two-fold increase of albumin expression compared to the original construct.
MATERIALS AND METHODS Bacteria and plasmids Plasmid constructions were routinely carried out in E. coli K-12, strain C600 galK (McKenney et al, 1981). After analysis and purification, plasmids were introduced in theE. coli B strain (Delbriick, 1946) for expression studies. Plasmid pXL276 has been described (Latta et al., 1987). Plasmids pDR720 (obtained from Pharmacia-Biochemicals) and pKRSlOl (Spindler et al, 1984) were used as sources of trp promoter regions. The trpR gene was obtained from pRPG9 (Gunsalus and Yanofsky, 1980). galK expression vectors are derivatives of pDS20 (Duester et al.,
[-77; +1] trp promoter. Plasmid pXL879 was obtained by ligating the =270-bp Pvu ll-Hpa I fragment of pKRSlOl carrying the [-260; +1] trp promoter to the large Eco Rl-Hpa I fragment of pXL534, blunt-ended with the the
Klenow enzyme. pXL712 was constructed by tandem insertion of the 80bp Eco RI fragment from pDR720 into the Eco RI site of pXL534. As in the case of pXL1313 (see below) and as already shown by Tacon et al. (1983), all the randomly analyzed resulting plasmids contained two or three promoter fragments, all of which were in the same orientation. Plasmids pXL717, pXL730, and pXL1077 were constructed by inserting the 1.3-kb Bam HI fragment of pRPG9 containing trpR under the control of its own operator/promoter into the single Bam HI site of pXL534, pXL712, and pXL879, respectively. The first A + T-rich block was chemically synthesized as two complementary oligonucleotides: 5'-AATAAATATTCTGAAAT-3' and 5'-AATTAAATATTCTGAAAT-3' and plasmids pXL1741, pXL1743, and pXL1745 were obtained by inserting the hybridized oligonucleotides into the filled-in Eco RI site of pXL305, pXL534, and pXL717, respectively. All plasmids carrying the different promoters were designed to have the same structure except for the promoter upstream regions. The trpR fragment was always inserted in the same orientation (same as HS A or galK).
1982). Construction
Construction
of the [-77, +1] Ptrp cassette
The Eco Rl-Sal I fragment containing region [-77;+ 1] of the trp promoter was chemically synthesized as the two following complementary oligonucleotides: 5'-AATTCGA-
CATCACAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCGCGAACTAGTTAACTAGTACGCAGCTTGGCTGCAGG-3' and 5'TCGACCTGCAGCCAAGCTGCGTACTAGTTAACTAGTTCGATGATTAATTGTCAACAGCTCATTTCAGAATATTTGCCAGAACCGTTGTGATGTCG-3'. These 95-mer oligonucleotides were synthesized using the Biosearch (Model 8600) automated DNA synthesizer utilizing
/3-cyanoethyl-protected phosphoramidite chemistry (Sinha et al., 1984). The nucleotide sequence of the promoter region was checked in the final expression constructs by "supercoil sequencing" (Chen and Seeburg, 1985). Construction
expression
of human
serum
albumin
vectors
Plasmid pXL534 was derived from pXL276 (Latta et al., 1987) by deleting a 2135-bp fragment, containing nones-
sential DNA and located 561 bp downstream from the stop codon of the albumin coding sequence, after the rrnB terminator region. A unique Bam HI site was inserted in that position. The 5.1-kb Exo Rl-Sal I fragment of pXL534 was ligated to the synthetic Eco Rl-Sal I [-77; + 1] trp promoter fragment described above to yield pXL670. In this vector, the expression of albumin is under the control of
of galK expression
vectors
All plasmids are derived from pDS20. The Eco Rl-Hind III fragment of pXL276 carrying the [-40;+ 1] trp promoter was inserted between the Eco RI and Hind III sites
of pDS20 to give pXL305 (Fig. 1). The [-77;+ 1] trp promoter cassette, synthesized as an Eco Rl-Sal I fragment, or the Eco Rl-Sal I fragment of pXL879 (see above) carrying the [-260;+ 1] trp promoter were inserted between the Eco RI and Sal I sites of
pXL305
to
give pXL814
or
pXL892, respectively.
Tandem insertion of the 80-bp Eco RI fragment of pDR720 containing the [-40;+ 1] promoter region into the Eco RI site of pXL305 gave pXL1312 and pXL1313, where galK is under the control of two or three repeating trp promoter
units, respectively.
Media and cell
growth
Bacteria were grown overnight at 37 °C in M9 synthetic medium (Miller, 1972) supplemented with 0.4% glucose, 0.4% casamino acids, and 10 fig of thiamine/ml, in presence of 100 fig of ampicillin/ml and 100 fig of L-tryptophan/ml. We chose a concentration of 0.4% casamino acids (Difco) over lower concentrations of casamino acids because we observed that regulation of the trp promoter was more efficient (not shown). The overnight culture was then diluted 100-fold either in the same medium plus (repression conditions) or minus (derepression conditions); only 40 fig of L-tryptophan/ml and bacteria were grown at 37°C for 5-6 hr until late exponential phase.
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MARTINE
LATTA, MICH\l=E`\LE PHILIT, ISABELLE MAURY, FABIENNE SOUBRIER, PATRICE DEN\l=E`\FLE, and JEAN-FRAN\l=C;\OIS MAYAUX
ABSTRACT A collection of variant plasmids expressing either Escherichia coli galactokinase or human serum albumin under the control of several E. coli trp promoter derivatives were constructed and studied for both efficiency of expression and regulation by tryptophan. Several variables, including the length of the upstream region, tandem duplications of a core promoter, and the insertion of the trp repressor trpR gene onto the expression vector, were studied. It is shown that derivatives containing sequences upstream from the \p=m-\35 region or multiple copies of the trp promoter produce twofold higher levels of protein than plasmids with a minimal trp promoter truncated at \p=m-\40. We show that the expression of a heterologous protein such as albumin can be significantly improved (13% vs. 7% of total proteins) if both the upstream trp promoter region, which enhances promoter strength, and an intact trpR gene, are included on the plasmids.
INTRODUCTION
(Landick and Yanofsky, 1987) and containing only the oppromoter elements (Harley and Reynolds, 1987). Although this 40-bp fragment contains all the sequence elements determining both the efficiency and regulation of transcriptional initiation (reviewed in Yanofsky et al, 1981), recent evidence indicates that elements outside the consensus core region might also be important for maximal transcription efficiency. For example, changing the first 20 nucleotides of the transcribed region may alter promoter strength 10-fold by influencing late events in transcriptional initiation (Kammerer et al, 1986). Comparison of strong E. coli promoters such as X PL, the lipoprotein (Ipp) or the Ptrp promoters indicated the presence of conserved A + T-rich blocks in the 5' upstream region (Nakamura and Inouye, 1979; Horn and Wells, 1981a); it has been proposed that these A + T-rich regions behave as constitutive transcriptional enhancers (Horn and Wells, 1981b; Nishi and Itoh, 1986). Poly(dA:dT) sequences have erator and core
(Ptrp) ÍS of the strongest regulated Escherichia coli promoters (Hallewell and Emtage, 1980; Russell and Bennett, 1982). It has often been used for the efficient expression of foreign proteins in E. coli (Nichols and Yanofsky, 1983; Tacon et al., 1983; Rosenberg et al, 1984). Recently, we reported that optimized expression vectors containing this promoter can be used in suitable E. coli strains to obtain high levels of intracytoplasmic expression of human proteins such as human serum albumin or tissue plasminogen activator (Latta et al, 1987; Sarmientos et al, 1989). Production of mature human interleukin 1/3 to several grams per liter of broth can be reached in high-cell density cultures of the E. coli B strain harboring such Ptrp expression plasmids (Jung et al, 1988). In particular, the nutritional requirements of wild-type strains such as E. coli B appeared more compatible with high-scale fermentation conditions at high biomass than those of current K12 laboratory strains. However, in these studies, we used a minimal Ptrp cassette (-39 to +1) lacking the attenuation control region
THE
PROMOTER OF THE TRYPTOPHAN OPERON
one
Laboratoire de
Génétique,
Institut de
also been shown to act as constitutive upstream promoter elements in yeast (reviewed in Struhl, 1986). To determine if transcriptional initiation from a tryptophan-regulated promoter can be improved further and to obtain higher production levels of heterologous proteins in
Biotechnologie, Rhône-Poulenc Santé,
94403
Vitry-sur-Seine Cedex, France.
E. coli from such promoters, we constructed several Ptrp derivatives containing either upstream regions of various lengths or multiple core promoter cassettes in tandem. We also studied the effect of the insertion of the trpR gene, coding for the Trp repressor, into the expression vectors. The in vivo strength and regulation of the different constructions were monitored both on the easily assayable E. coli galK gene and on the expression of heterologous HSA. Based on these results, we describe a new Ptrp vector that results in a stable two-fold increase of albumin expression compared to the original construct.
MATERIALS AND METHODS Bacteria and plasmids Plasmid constructions were routinely carried out in E. coli K-12, strain C600 galK (McKenney et al, 1981). After analysis and purification, plasmids were introduced in theE. coli B strain (Delbriick, 1946) for expression studies. Plasmid pXL276 has been described (Latta et al., 1987). Plasmids pDR720 (obtained from Pharmacia-Biochemicals) and pKRSlOl (Spindler et al, 1984) were used as sources of trp promoter regions. The trpR gene was obtained from pRPG9 (Gunsalus and Yanofsky, 1980). galK expression vectors are derivatives of pDS20 (Duester et al.,
[-77; +1] trp promoter. Plasmid pXL879 was obtained by ligating the =270-bp Pvu ll-Hpa I fragment of pKRSlOl carrying the [-260; +1] trp promoter to the large Eco Rl-Hpa I fragment of pXL534, blunt-ended with the the
Klenow enzyme. pXL712 was constructed by tandem insertion of the 80bp Eco RI fragment from pDR720 into the Eco RI site of pXL534. As in the case of pXL1313 (see below) and as already shown by Tacon et al. (1983), all the randomly analyzed resulting plasmids contained two or three promoter fragments, all of which were in the same orientation. Plasmids pXL717, pXL730, and pXL1077 were constructed by inserting the 1.3-kb Bam HI fragment of pRPG9 containing trpR under the control of its own operator/promoter into the single Bam HI site of pXL534, pXL712, and pXL879, respectively. The first A + T-rich block was chemically synthesized as two complementary oligonucleotides: 5'-AATAAATATTCTGAAAT-3' and 5'-AATTAAATATTCTGAAAT-3' and plasmids pXL1741, pXL1743, and pXL1745 were obtained by inserting the hybridized oligonucleotides into the filled-in Eco RI site of pXL305, pXL534, and pXL717, respectively. All plasmids carrying the different promoters were designed to have the same structure except for the promoter upstream regions. The trpR fragment was always inserted in the same orientation (same as HS A or galK).
1982). Construction
Construction
of the [-77, +1] Ptrp cassette
The Eco Rl-Sal I fragment containing region [-77;+ 1] of the trp promoter was chemically synthesized as the two following complementary oligonucleotides: 5'-AATTCGA-
CATCACAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCGCGAACTAGTTAACTAGTACGCAGCTTGGCTGCAGG-3' and 5'TCGACCTGCAGCCAAGCTGCGTACTAGTTAACTAGTTCGATGATTAATTGTCAACAGCTCATTTCAGAATATTTGCCAGAACCGTTGTGATGTCG-3'. These 95-mer oligonucleotides were synthesized using the Biosearch (Model 8600) automated DNA synthesizer utilizing
/3-cyanoethyl-protected phosphoramidite chemistry (Sinha et al., 1984). The nucleotide sequence of the promoter region was checked in the final expression constructs by "supercoil sequencing" (Chen and Seeburg, 1985). Construction
expression
of human
serum
albumin
vectors
Plasmid pXL534 was derived from pXL276 (Latta et al., 1987) by deleting a 2135-bp fragment, containing nones-
sential DNA and located 561 bp downstream from the stop codon of the albumin coding sequence, after the rrnB terminator region. A unique Bam HI site was inserted in that position. The 5.1-kb Exo Rl-Sal I fragment of pXL534 was ligated to the synthetic Eco Rl-Sal I [-77; + 1] trp promoter fragment described above to yield pXL670. In this vector, the expression of albumin is under the control of
of galK expression
vectors
All plasmids are derived from pDS20. The Eco Rl-Hind III fragment of pXL276 carrying the [-40;+ 1] trp promoter was inserted between the Eco RI and Hind III sites
of pDS20 to give pXL305 (Fig. 1). The [-77;+ 1] trp promoter cassette, synthesized as an Eco Rl-Sal I fragment, or the Eco Rl-Sal I fragment of pXL879 (see above) carrying the [-260;+ 1] trp promoter were inserted between the Eco RI and Sal I sites of
pXL305
to
give pXL814
or
pXL892, respectively.
Tandem insertion of the 80-bp Eco RI fragment of pDR720 containing the [-40;+ 1] promoter region into the Eco RI site of pXL305 gave pXL1312 and pXL1313, where galK is under the control of two or three repeating trp promoter
units, respectively.
Media and cell
growth
Bacteria were grown overnight at 37 °C in M9 synthetic medium (Miller, 1972) supplemented with 0.4% glucose, 0.4% casamino acids, and 10 fig of thiamine/ml, in presence of 100 fig of ampicillin/ml and 100 fig of L-tryptophan/ml. We chose a concentration of 0.4% casamino acids (Difco) over lower concentrations of casamino acids because we observed that regulation of the trp promoter was more efficient (not shown). The overnight culture was then diluted 100-fold either in the same medium plus (repression conditions) or minus (derepression conditions); only 40 fig of L-tryptophan/ml and bacteria were grown at 37°C for 5-6 hr until late exponential phase.
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