Stringent regulation and high-level expression of heterologous genes in Escherichia coli using T7 system controllable by the araBAD promoter

The recombinant Eschreichia coli strain BL21 (BAD) was constructed to carry a chromosomal copy of T7 gene 1 fused to the araBAD promoter. To further characterize this expression system, strain BL21 (BAD) was transformed with the plasmid containing the carbamoylase gene from Agrobacterium radiobacter driven by the T7 promoter. Upon induction with L-arabinose, recombinant cells produced 100-fold increase in carbamoylase activity in comparison with uninduced cells on M9 semidefined medium plus glycerol. This protein yield accounts for 30% of total cell protein content. In addition, it was found that after 100 generations the plasmid harboring the carbamoylase gene remained firmly stable in strain BL21 (BAD), but its stability dropped to only 20-30% in strain BL21 (DE3), a commercial strain bearing T7 gene 1 regulated by the lacUV5 promoter in its chromosome. In an attempt to enhance the total protein yield, fed-batch fermentation process was carried out using a two-stage feeding strategy to compartmentalize cell growth and protein synthesis. In the batch fermentation stage, the culture was grown on glucose to reach the stationary growth phase. Subsequently, glycerol was fed to the culture broth and L-arabinose was augmented to induce protein production when cells entered the late log growth phase. As a result, a carbamoylase yield corresponding to 5525 units was obtained, which amounts to a 337-fold increase over that achieved on a shake-flask scale. Taken together, these results illustrate the practical usefulness of T7 system under control of the araBAD promoter for heterologous protein production.     

Development of a fed-batch fermentation process to overproduce phosphoenolpyruvate carboxykinase using an expression vector with promoter and plasmid copy number controllable by heat

To effectively achieve tight regulation and high-level expression of cloned genes, a novel expression plasmid has been developed to contain the promoter and allow the plasmid copy number to be controlled by heat. The feasibility of the plasmid was tested by overproducing the pck gene product (Pck), a protein responsible for cell growth on gluconeogenic carbons and with potential toxicity. By fusing the pck gene with the promoter on the plasmid, the Escherichia coli strain harboring the composite vector was shown to produce various amounts of Pck in response to different degrees of heat shock. With the use of a 30 degrees -->41 degrees C stepwise upshift, the shake-flask culture of recombinant cells enabled production of maximal Pck in soluble form accounting for 20% of total cell protein. In sharp contrast, Pck production was undetectable in the uninduced cell, and this was further confirmed by the failed growth of strain JCL1305, defective in the essential genes for gluconeogenesis, carrying the composite vector on succinate at 30 degrees C. By exploiting the fed-batch fermentation approach, the recombinant cell batch initially kept at 30 degrees C in a lab-scale fermentor was exposed to 41 degrees C for 2 h at the batch fermentation stage, followed by a reduction in temperature to 37 degrees C throughout the remainder of the culturing process. Consequently, this resulted in Pck production equivalent to 15% of total cell protein. The total Pck yield thus calculated was amplified 1880-fold over that obtained at the shake-flask scale. Overall, there is great promise for this expression system due to its tight control, high production, simple thermomodulation, and feasible scale-up of recombinant proteins.     

A facile and efficient method to achieve LacZ overproduction by the expression vector carrying the thermoregulated promoter and plasmid copy number

On the basis of the runaway-replication vector, an expression plasmid was developed to achieve tight regulation as well as high-level expression of cloned genes by thermal control of the promoter together with the plasmid copy number. To demonstrate the feasibility of this approach, the lacZ gene was fused with the heat-inducible promoter on the vector, and the result showed that protein production levels in the Escherichia coli strain harboring the recombinant plasmid could be varied in response to various degrees of heat shock. The maximal soluble LacZ ranging between 45 000 and 50 000 Miller units was obtained as the recombinant strain received a 30 --> 40 degrees C stepwise upshift, and it accounted for a 450-fold amplification over an uninduced level. Further analyses by SDS-PAGE indicated the maximal protein production (including soluble and insoluble forms) in the bacteria reaching approximately 30% total cell protein. In addition, two approaches were demonstrated to be very useful in enhancing the total soluble LacZ production on a fermenter scale. One was to shuttle the culture between two fermenters connected in series and set at different temperatures. The other resorted to the use of two-step temperature alteration in a batch fermenter, namely, raising the temperature to 40 degrees C for a certain period of time followed by reducing the temperature to 37 degrees C. Overall, it illustrates the remarkable features of the expression system with stringent regulation, high-level production capacity, facile induction, and high stability, and the usefulness of this system for recombinant protein productions is promising.     

Improvement of the thermoregulated T7 expression system by using the heat-sensitive lacI

The thermoregulated T7 expression system was previously reported to be an effective way to produce massive amounts of recombinant proteins (Chao, Y. P.; Law, W. S.; Chen, P. T.; Hung, W. B. High production of heterologous proteins in Escherichia coli using the thermo-regulated T7 expression system. Appl. Microbiol. Biotechnol. 2002b, 58, 446-453). To ensure its practical applicability, the system was improved for stringency with the construction of the T7lac-promoter-containing plasmid associated with the thermolabile lacI gene (lacIts). Owing to the recessive feature of lacIts, the wild-type lacI was removed from the genome of the cell. Moreover, the cell was engineered to carry the chromosomal copy of the T7 gene 1 subject to the regulation of lambdaPL and lambdaPR promoters. To characterize the system, the lacZ gene was fused to the T7lac promoter, and subsequent experiments showed that various amounts of LacZ could be synthesized in the plasmid-bearing cell in response to heat. Among the producers, the cell with the plasmid containing lacIts (substitution of Gly265 with Asp in lacI) was able to produce the maximal LacZ, the production accounting for an amplification of more than 200-fold over the uninduced level. A further demonstration was carried out to illustrate the practical usefulness of the developed system by producing carbamoylase on a 4000 L scale. Cultured to reach high cell density, the carbamolyase-producing cell was shown to retain plasmids with 95% stability and to be capable of producing soluble protein equal to 13% of the total cell proteins. Overall, it illustrates the remarkable features of the developed system with tightness, high expression level, thermal inducibility, and high stability.     

Coupling the T7 A1 promoter to the runaway-replication vector as an efficient method for stringent control and high-level expression of lacZ

An expression vector characterized by tight regulation and high expression of cloned genes appears to be indispensable for the engineering need. To achieve this goal, in association with lacI the T7 A1 promoter containing two synthetic lac operators was constructed into a runaway-replication vector. To further examine this vector system, lacZ was subcloned and placed under the control of the T7 A1 promoter on the plasmid. With the application of the thermal induction alone, the Escherichia coli strain harboring the recombinant plasmid was able to produce 15,000 Miller units of beta-galactosidase, while it yielded the recombinant protein with 45,000-50,000 Miller units upon both thermal and chemical induction. In sharp contrast, only 60-90 Miller units of beta-galactosidase was obtained for the cell at an uninduced state. As a result, the production yield of beta-galactosidase over the background level is amplified approximately 170-fold by thermal induction and 500-fold by thermal and chemical induction. To produce the recombinant protein on a large scale, an approach by connecting two fermenters in series was newly developed. By applying the three-stage temperature shift in this dual fermenter system, 55,000 Miller units of beta-galactosidase was obtained. Overall, it shows the potential use of the vector system developed here for its tight control and high production of recombinant proteins.     

Construction of a low-temperature protein expression system using a cold-adapted bacterium, Shewanella sp. strain Ac10, as the host

A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4 degrees C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce beta-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4 degrees C and 139 mg/liter of culture at 18 degrees C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium, Desulfotalea psychrophila DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system.     

节杆菌BT801 N-氨甲酰氨基酸水解酶基因的克隆与表达

通过PCR从质粒pUC18 16 9中扩增得到N 氨甲酰氨基酸水解酶基因 (hyuC) ,置于原核表达载体pQE6 0的T5启动子下游构成表达质粒pQE6 0 hyuC ,并在大肠杆菌M15中实现了该基因的高表达。SDS PAGE检测表达产物 ,在相对分子量 44kD处有一表达带 ,经薄层扫描分析目的蛋白占全菌蛋白的 40 % ,主要以可溶性形式存在。酶活性分析结果表明 ,工程菌M15 pQE6 0 hyuC的N 氨甲酰氨基酸水解酶的比活分别比原始菌株ArthrobacterBT80 1和亚克隆DH5α pUC18 16 9提高了 5 2倍和 72倍。在节杆菌BT80 1和大肠杆菌DH5α pUC18 16 9的反应体系中加入等量菌体的工程菌M15 pQE6 0 hyuC ,可使乙内酰脲酶总比活分别提高 8 1倍和 3 0倍。     

One-step production of D-p-hydroxyphenylglycine by recombinant Escherichia coli strains

The gene encoding D-hydantoinase from Agrobacterium radiobacter NRRL B11291 was successfully cloned by use of polymerase chain reaction. A positive clone was scored, and its nucleotide sequence was further analyzed. The analysis by deleting various lengths of nucleotides from the amino terminus of the open reading frame revealed the putative regions for promoter and RBS site. By highly expressing both D-hydantoinase and carbamoylase, recombinant Escherichia coli strains were able to convert DL-hydroxyphenyl hydantoin (DL-HPH) to D-p-hydroxyphenylglycine (D-HPG) with a conversion yield of 97%, accounting for productivity 5 times higher than that obtained by A. radiobacter NRRL B11291. Immobilizing the recombinant cells with kappa-carrageenan could also achieve a conversion of 93%, while A. radiobacter NRRL B11291 attained 20% within the same period of reaction time. These results illustrate the feasibility in employing recombinant E. coli to accomplish one-step conversion of DL-HPH to D-HPG. In the process of improving D-HPG production, D-hydantoinase activity was increased 2.57-fold but carbamoylase activity remained constant, which resulted in only a 30% increase in the reaction rate. It suggests that carbamoylase is the step setting the pace of the reaction. Since the reaction substrate is highly insoluble, achieving sufficient agitation appears to be an important issue in this heterogeneous system. This view is further supported by the study on repeated use of cells, which shows that to reach a conversion of more than 90% free cells can be recycled six times, whereas immobilized cells can be used only twice. In conclusion, the poor reusability of immobilized cells is due to the fouling on the gel surface.     

Fluorescence based assay of GAL system in yeast Saccharomyces cerevisiae

The GAL1 promoter is one of the strongest inducible promoters in the yeast Saccharomyces cerevisiae. In order to improve recombinant protein production we have developed a fluorescence based method for screening and evaluating the contribution of various gene deletions to protein expression from the GAL1 promoter. The level of protein synthesis was determined in 28 selected mutant strains simultaneously, by direct measurement of fluorescence in living cells using a microplate reader. The highest, 2.4-fold increase in GFP production was observed in a gal1 mutant strain. Deletion of GAL80 caused a 1.3-fold increase in fluorescence relative to the isogenic strain. GAL3, GAL4 and MTH1 gene deletion completely abrogated GFP synthesis. Growth of gal7, gal10 and gal3 also exhibited reduced fitness in galactose medium. Other genetic perturbations affected the GFP expression level only moderately. The fluorescence based method proved to be useful for screening genes involved in GAL1 promoter regulation and provides insight into more efficient manipulation of the GAL system.     

Single-step purification and structural characterization of human interleukin-6 produced in Escherichia coli from a T7 RNA polymerase expression vector

Human interleukin-6 or B-cell stimulatory factor-2 is a cytokine involved in acute phase and immune response. Cloning of cDNA for human interleukin-6 in the pT7.7 expression plasmid under the control of a bacteriophage T7 RNA polymerase promoter system allows rapid production of the cytokine in Escherichia coli. Upon cell induction with isopropyl thiogalactopyranoside, recombinant human interleukin-6 is overexpressed and forms insoluble inclusion bodies. Solubilization of the protein with 6 M guanidine hydrochloride and refolding in the presence of a reduction/oxidation system results in a quantitative recovery of recombinant human interleukin-6. This material is already 70% pure and can be further purified to homogeneity with a single passage over a weak anionic-exchange column. Extended structural characterization of the purified protein by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry demonstrates that recombinant human interleukin-6 is identical to the natural protein both in amino acid sequence and S-S bridge content. However, it contains a minor component accounting for about 20% of the entire translated protein which exhibits a Met-Ala dipeptide extension at the N-terminus. Purified recombinant human interleukin-6 is biologically active because it is able to induce at least 70-fold the human C-reactive promoter transfected in human hepatoma Hep 3B cells and is stable for several months in 10% glycerol at 4 degrees C. The expression system described in the present work has the main advantage of producing a high yield of recombinant human interleukin-6 (about 25 mg/l) combined with a very simple purification scheme.     

Low temperature cultivation of Escherichia coli carrying a rice lipoxygenase L-2 cDNA produces a soluble and active enzyme at a high level

Using a T7 RNA polymerase promoter system, rice lipoxygenase L-2 cDNA was expressed in E. coli as a fusion protein with 18 amino acid residues at the amino terminal end of the original enzyme. Incubation at 37 degrees C for 3 h in the presence of the inducer resulted in the production of inactive lipoxygenase. However, when induction was carried out at 15 degrees C for 16 h, active lipoxygenase, amounting to 3% of the total soluble protein, was produced. The enzyme was purified by ammonium sulfate precipitation and Mono-Q column chromatography to homogeneity at a yield of 80%. Expression of this protein should permit future site-directed mutagenesis of the gene and crystallization of the enzyme.     

Induction of T4 DNA ligase in a recombinant strain of Escherichia coli

The kinetics of cell growth and foreign protein production, as well as factors affecting protein stability, were studied and optimized in batch and fed-batch fermentations of a recombinant strain of Escherichia coli. The pL promoter from bacteriophage lambda under the control of a temperature-sensitive cl represser, with the entire construct integrated into the E. coli chromosome through the use of a defective bacteriophage lambda lysogen, was used to direct the synthesis of T4 DNA ligase. The biphasic fermentations consisted of a primary growth phase at 30 degrees C followed by an induction phase which was initiated by shifting the temperature to 42 degrees C. In the fed-batch fermentations, additional nutrients were added at the time of initiating induction. Maintenance of sufficiently high concentrations of the organic substrates (glucose and casamino acids) during the induction phase was required for continued cell growth at 42 degrees C. Such growth was essential for T4 DNA ligase formation and in vivo stability. Hence, fed-batch fermentations produced the highest yield of the foreign protein Commensurate with providing lower total amounts of substrates. In such cases, high cell densities (6 g dry wt/L) with substantial intracellular levels of T4 DNA ligase (4.6% total cellular protein, or 2.7% of the dry biomass) were achieved.     

Construction and characterization of thermo-inducible vectors derived from heat-sensitive lacI genes in combination with the T7 A1 promoter

The lack of stringency and the cost of induction are two major disadvantages of using lac-derived vectors for recombinant protein productions. To compensate for these drawbacks, a series of thermo-inducible vectors was developed by coupling heat-sensitive lacI (lacIts) with the T7 A1 promoter on a multiple-copynumber plasmid. The lacIts genes were created by the introduction of Gly187-->Ser substitution along with three alternative mutation sites, Leu233-->Lys, Ala241-->Thr, and Gly265-->Asp, generated by site-directed mutagenesis into the wild-type lacI gene. With the LacZ production as a model, the induction profiles for various vectors containing distinct lacIts exhibited a positive trend as the temperature increased. The fully induced level was achieved by applying the temperature shift from 30 degrees C to 42, 40, or 37 degrees C to the cells harboring the plasmid with the Gly187-->Ser, Ala241-->Thr, or Gly265-->Asp substitution in lacI, respectively. As a result, it produced the maximal LacZ production ranging between 46,000 and 54,000 Miller units, corresponding to a 100- to 400-fold amplification over the uninduced level. As a whole, these novel expression vectors are characterized as having tight regulation and facile inducibility, and their practical usefulness in industrial production of recombinant proteins appears promising.     

Induction of the heat shock regulon of Escherichia coli markedly increases production of bacterial viruses at high temperatures.

Production of bacteriophages T2, T4, and T6 at 42.8 to 44 degrees C was increased from 8- to 260-fold by adapting the Escherichia coli host (grown at 30 degrees C) to growth at the high temperature for 8 min before infection; this increase was abolished if the host htpR (rpoH) gene was inactive. Others have shown that the htpR protein increases or activates the synthesis of at least 17 E. coli heat shock proteins upon raising the growth temperature above a certain level. At 43.8 to 44 degrees C in T4-infected, unadapted cells, the rates of RNA, DNA, and protein synthesis were about 100, 70, and 70%, respectively, of those in T4-infected, adapted cells. Production of the major processed capsid protein, gp23, was reduced significantly more than that of most other T4 proteins in unadapted cells relative to adapted cells. Only 4.6% of the T4 DNA made in unadapted cells was resistant to micrococcal nuclease, versus 50% in adapted cells. Thus, defective maturation of T4 heads appears to explain the failure of phage production in unadapted cells. Overproduction of the heat shock protein GroEL from plasmids restored T4 production in unadapted cells to about 50% of that seen in adapted cells. T4-infected, adapted E. coli B at around 44 degrees C exhibited a partial tryptophan deficiency; this correlated with reduced uptake of uracil that is probably caused by partial induction of stringency. Production of bacteriophage T7 at 44 degrees C was increased two- to fourfold by adapting the host to 44 degrees C before infection; evidence against involvement of the htpR (rpoH) gene is presented. This work and recent work with bacteriophage lambda (C. Waghorne and C.R. Fuerst, Virology 141:51-64, 1985) appear to represent the first demonstrations for any virus that expression of the heat shock regulon of a host is necessary for virus production at high temperature.     

Effect of ethanol and low-temperature culture on expression of soybean lipoxygenase L-1 in Escherichia coli.

We have constructed a full-length cDNA that encodes soybean seed lipoxygenase L-1 and have expressed it in Escherichia coli. This gene was inserted into a pT7-7 expression vector, containing the T7 RNA polymerase promoter. E. coli, strain BL21 (DE3), which carries the T7 promoter in its genome, was transfected with the plasmid. Expression of this gene when the cells were cultured at 37 degrees C yielded polypeptide that was recognized by anti-L-1 antibody, but had very little lipoxygenase activity. Yields of active enzyme were markedly increased when cells were cultured at 15-20 degrees C. When ethanol, which has been reported to be an excellent elicitor of heat-shock proteins in E. coli, was also present at a level of 3% the yield was further increased by 40%. Under optimum conditions 22-30 mg of soluble active enzyme was obtained per liter of culture.     

Cloning of lysozyme and lysostaphin genes of Staphylococcus aureus and their expression in Bacillus subtilis cells

The gene of microbial lysozyme (lyz) of S. aureus 118 and the gene of lysostaphin (lzf) of S. aureus RN 3239 were cloned and their expression in B. subtilis cells was shown. Lysozyme production in B. subtilis recombinant clone pLF14-Lyz, obtained as the result of cloning, was 2.5-fold greater than lysozyme production in S. aureus wild strain 118. Lysostaphin production in B. subtilis recombinant strain pLF14-Lzf which had inherited the cloned genes was approximately equal to lysostaphin production observed in S. aureus initial strain RN 3239. The production of lysozyme and lysostaphin in the cells of B. subtilis recombinant strains was observed at 30 degrees C and pH 5.5, while in S. aureus initial strains 118 and RN 3239 bacteria produced lysozyme and lysostaphin at 37 degrees C and pH 7.5 respectively.