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.     

High-level expression of a<Emphasis Type="Italic"> lacZ</Emphasis> gene from a bacterial artificial chromosome in<Emphasis Type="Italic"> Escherichia coli</Emphasis>

The GlnAP2 element has been proved to be an effective and inducible-by exogenous acetate-promoter in Escherichia coli with glnL/pta double mutations. Based on this feature, a single-copy expression vector was constructed via coupling of the glnAP2 promoter-regulated T7 RNA polymerase gene and the T7-promoter-controlled lacZ gene on a bacterial artificial chromosome. After induction with 20 mM potassium acetate, the glnL/pta double mutant E. coli harboring the single-copy plasmid produced 47,500 Miller units of beta-galactosidase activity. This high level expression, corresponding to 27% of total cell protein, was comparable to that determined with the commercial multi-copy expression vector, pET-14b, in strain E. coli Tuner (DE3) (64,300 Miller units, 41% of total cell protein). Moreover, this single-copy expression vector could be maintained for at least 150 generations even in the presence of inducers. In contrast, the multi-copy expression vector was extensively lost after induction. The results indicate that the single-copy expression system has the potential for high-level heterologous protein production for industrial applications.     

Enhancing recombinant protein yields in Escherichia coli using the T7 system under the control of heat inducible lambda PL promoter

A recombinant plasmid containing the complete lacZ gene downstream of the T7 promoter was used to transform Escherichia coli containing another plasmid which had the T7 RNA polymerase gene under the control of heat inducible lambda PL promoter. This recombinant E. coli containing the two plasmids was studied in order to enhance beta-galactosidase expression. The heat shock time which effectively regulates the T7 RNA polymerase was optimized and best expression of beta-galactosidase was obtained with 2 min heat shock. Substrate feeding increased the duration of log phase and allowed induction at a higher cell density without affecting the specific activity. A high cell density (7 g l-1) and high specific activity (approximately 20,000 U) were achieved which effectively increased the product concentration 18-fold.     

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.     

Fed-batch cultivation of an oxygen-dependent inducible promoter system, the nar promoter in Escherichia coli with an inactivated nar operon

The nar promoter of Escherichia coli is maximally induced under anaerobic or microaerobic conditions in the presence of nitrate. We previously demonstrated in batch experiments that the intact nar promoter of E. coli cloned into a pBR322-based plasmid serves as a high-level expression system in a nar mutant of E. coli (Lee et al., 1996b). In this study, we extend characterization of the nar promoter expression system to the fed-batch culture mode, which is widely used in industrial-scale fermentation. From these experiments, it was found that the specific beta-galactosidase activity expressed from the lacZ gene fused to the nar promoter was maximal when host cells were grown under aerobic conditions [dissolved oxygen, (DO) = 80%] to absorbance at 600 nm (OD600) = 35 before induction of the nar promoter by lowering DO to 1-2% with alternating microaerobic and aerobic conditions. Approximately 15 h after induction, the OD600 of the culture reached 135 and the specific beta-galactosidase activity increased to 40,000 Miller units, equivalent to approximately 35% of the total cellular proteins. The specific beta-galactosidase activity before induction was approximately 1,000 Miller units, giving an induction ratio of approximately 40. Based on these results, we conclude that the nar promoter provides a convenient and effective high level expression system under conditions of fed-batch culture.     

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.     

Characterization of an oxygen-dependent inducible promoter system, the nar promoter, and Escherichia coli with an inactivated nar operon

The nar promoter of Escherichia coli, which is maximally induced under anaerobic conditions in the presence of nitrate, was characterized to see whether the nar promoter cloned onto pBR322 can be used as an inducible promoter. To increase the expression level, the nar promoter was expressed in E. coli where active nitrate reductase cannot be expressed from the nar operon on the chromosome. A plasmid with the lacZ gene expressing beta-galactosidase instead of the structural genes of the nar operon was used to simplify an assay of induction of the nar promoter. The following effects were investigated to find optimal conditions: methods of inducing the nar promoter, optimal nitrate and molybdate concentrations maximally inducing the nar promoter, the amount of expressed beta-galactosidase, and induction ratio (specific beta-galactosidase activity after maximal induction/specific beta-galactosidase activity before induction.)The following results were obtained from the experiments: induction of the nar promoter was optimal when E. coli was grown in the presence of 1% nitrate at the beginning of culture; expression of beta-galactosidase was not affected by molybdate; the induction ratio was maximal, approximately 300, when the overnight culture was grown in the flask for 2.5 h (OD(600) is congruent to 1.3) before being transferred to the fermentor; the amount of beta-galactosidase per cell and per medium volume was maximal when E. coli was grown under aerobic conditions to OD(600) = 1.7; then the nar promoter was induced under microaerobic conditions made by lowering dissolved oxygen level (DO) to 1-2%. After approximately 6 h of induction, OD(600) became 3.2 and specific beta-galactosidase activity became 36,000 Miller units, equivalent to 35% of total cellular proteins, which was confirmed from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (c) 1996 John Wiley & Sons, Inc.     

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.     

Influence of plasmid origin and promoter strength in fermentations of recombinant yeast

The effects of plasmid promoter strength and origin of replication on cloned gene expression in recombinant Saccharomyces cerevisiae have been studied in batch and continuous culture. The plasmids employed contain the Escherichia coli lacZ gene under the control of yeast promoters regulated by the galactose regulatory circuit. The synthesis of beta-galactosidase was therefore induced by the addition of galactose. The initial induction transients in batch culture were compared for strains containing plasmids with 2mu and ARS1 origins. As expected, cloned gene product synthesis was much lower with the ARS1 plasmid: average beta-galactosidase specific activity was an order of magnitude below that with the 2mu-based plasmid. This was primarily due to the low plasmid stability of 7.5% when the plasmid origin of replication was the ARS1 element. The influence of plasmid promoter strength was studied using the yeast GAL1, GAL10, and hybrid GAL10-CYC1 promoters. The rate of increase in beta-galactosidase specific activity after induction in batch culture was 3-5 times higher with the GAL1 promoter. Growth rate under induced conditions, however, was 15% lower than in the absence of lacZ expression for this promoter system. The influence of plasmid promoter strength on induction behavior and cloned gene expression was also studied in continuous fermentations. Higher beta-galactosidase production and lower biomass concentration and plasmid stability were observed for the strain bearing the plasmid with the stronger GAL1 promoter. Despite the decrease in biomass concentration and plasmid stability, overall productivity in continuous culture using the GAL1 promoter was three times that obtained with the GAL10-CYC1 promoter.     

Generation of an AraC-araBAD promoter-regulated T7 expression system

An alternative and facile delivery system for T7 RNA polymerase has been devised and constructed. T7 gene 1 has been placed under control of the araBAD promoter element regulated by the AraC protein. Cotransformation of the resultant plasmid, pTara, with one containing a target gene under T7 promoter-regulated expression potentially allows repression by glucose and induction by arabinose in the range of 0.5 to 20 mM sugar concentration. To demonstrate the efficacy of this expression system, the p53 gene under T7 promoter control in two different plasmids was expressed in Escherichia coli using pTara as the source of T7 RNA polymerase. Repression and induction of p53 were achieved in both a lower and higher copy number plasmid, although the levels of induction were higher with the lower copy number expression vector. Cotransformation of an expression plasmid with pTara provides a low-cost method of T7 RNA polymerase-regulated expression that can be fine-tuned using glucose and arabinose concentrations to balance protein expression with potential solubility or toxicity problems.     

Coexpression of the type I signal peptidase gene sipM increases recombinant protein production and export in Bacillus megaterium MS941

The removal of the signal peptide from a precursor protein is a crucial step of protein secretion. In order to improve Bacillus megaterium as protein production and secretion host, the influence of homologous type I signal peptidase SipM overproduction on recombinant Leuconostoc mesenteroides dextransucrase DsrS synthesis and export was investigated. The dsrS gene was integrated as a single copy into the chromosomal bgaM locus encoding beta-galactosidase. Desired clones were identified by blue-white selection. In this strain, the expression of sipM from a multicopy plasmid using its own promoter increased the amount of secreted DsrS 3.7-fold. This increase in protein secretion by SipM overproduction was next transferred to a high level DsrS production strain using a multicopy plasmid encoding sipM with its natural promoter and dsrS under control of a strong xylose-inducible promoter. No further increase in DsrS export were observed when this vector was carrying two sipM copies. Similarly, bicistronic sipM and dsrS high level expression did not enhance DsrS secretion, indicating the natural limitation of the approach. Interestingly, SipM-enhanced DsrS secretion also resulted in an overall increase of DsrS production.     

Overexpression of cloned genes using recombinant Escherichia coli regulated by a T7 promoter: I. Batch cultures and kinetic modeling

A novel Eschericha coli expression system directed by bacteriophage T7 RNA Polymerase utilized for overexpression of the cloned gene. The recombinant cell contains the plasmid with a bacteriophage promoter, the T7 promoter, to regulate the expression of the target gene. This promoter is recongnized only by T7 RNA polymerase, whose gene has been fused into the host chromosome and is under control of the lacUV5 promoter. Therefore, the target gene on the plasmid can be expressed only in the presence of T7 RNA polymerase, which is induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The batch cultures were performed to investigate the effect of induction on kinetics of cell growth and foreign protein formation and to determine the optimal induction strategy. It was observed that the specific growth rates of the recombinant cells dramatically decrease after induction, and that there is an optimal induction time for maximizing the accumulated intracellular foreign protein. This optimal induction time varies singificantly with inducer concentration. To better understand the optimal behavior, a lumped mechanistic model was constructed to analyze the induced cell growth and foreign protein formation rates. (c) 1992 John Wiley & Sons, Inc.     

A comparison of mathematical model predictions to experimental measurements for growth and recombinant protein production in induced cultures of Escherichia coli

Recombinant cell growth and protein synthesis by a recombinant Escherichia coli under various inducing conditions are compared to the predictions of a mathematical model. The mathematical model used was a combination of two literature models: (1) an empirical kinetic model for recombinant growth and product formation and (2) a genetically structured model of the lac promoter-operator on a multicopy plasmid. The experimental system utilized was recombinant E. coli CSH22 bearing the temperature-sensitive plasmid pVH106/172, which codes for the synthesis of beta-galactosidase and the other lac operon genes under the control of a lac promoter. Mathematical model predictions for recombinant beta-galactosidase yield and specific growth rate were compared with fermentation measurements of these same quantities for conditions of chemical induction with cyclic AMP and IPTG, copy number amplification (by shifting culture temperature), and combined chemical induction and copy number amplification. The model successfully predicted experimental product yields for most cases of chemical induction even though the product yields varied from 0.34 x 10(3) to 1500 x 10(3) units/g cell mass. The kinetic model also correctly predicted a decline in the specific growth rate with increasing levels of plasmid and recombinant protein. The model was less successful at predicting product amplification at high copy numbers. A comparison of model predictions and experimental results was also used to investigate some of the assumptions used in constructing the mathematical models.     

Applicability of new expression vectors for both engineering uses and biological studies

To be applicable for both engineering and biological uses, the plasmid with the features of tight regulation, high-level expression, and subtle modulation (or homogeneous induction) is required. IPTG-inducible promoters are of particular interest since they acquire the latter two merits but usually lack stringency. To this end, two plasmids have been developed to contain the T7 A1 promoter along with either lacI(q) or lacI gene. As a production system, the cells harboring the plasmids with the lacZ gene clone enabled production of the maximal protein accounting for 35% total cell content upon induction by a saturating IPTG level. This protein yield is amplified over 700-fold relative to that at the uninduced state. As a system for biological study, the ppc negative strain bearing the plasmid with the ppc gene clone failed to grow on glucose without IPTG induction but immediately resumed its growth in the presence of IPTG. Moreover, the level of the ppc gene product in the cell was varied by various IPTG, and the result revealed that the wild-type ppc level was sufficient to support the saturated growth of the cell on glucose. Overall, it illustrates the applicability of these plasmids to needs in the post-genome era.     

High production of heterologous proteins in Escherichia coli using the thermo-regulated T7 expression system

The exclusive use of isopropyl beta-D-thiogalactopyranoside to activate the T7 promoter for protein production has limited the general use of the expression system. We have sought an alternative by constructing a recombinant Escherichia coli strain, BL21 (G2), to carry a chromosomal copy of T7 gene 1 fused to the lambdaPL and lambdaP(R) tandem promoter. As a result, the recombinant strain harboring the carbamoylase gene from Agrobacterium radiobacter NRRL B11291 was shown to display various levels of.protein production in response to different degrees of heat shock. In particular, the system remained inactive at 30 degrees C and exhibited high sensitivity to heat such that a detectable carbamoylase activity could be measured after exposure to 33 degrees C. Moreover, heating in two steps - elevating the temperature from 30 degrees C to 39 degrees C and holding for a brief period, followed by reducing to 37 degrees C--was found to be the most potent method for protein production in this case. Using this approach, the recombinant protein accounted for 20% of total protein content of the cell. These results reveal the advantages of this expression system: responsiveness to thermal modulation and high-level production capability. In an attempt to enhance the total protein yield, a fed-batch fermentation process was carried out to control the cell growth rate by adjusting the substrate inflow. By applying the two-step temperature change. a carbamoylase yield with enzyme activity corresponding to 14,256 units was obtained. This production yield is a 10-fold increase in comparison with that at the batch-fermentation scale and 2,000-fold higher than that achieved at the shake-flask scale. Overall, it illustrates the promise of the newly constructed T7 system based on heat inducibility for industrial scale production of recombinant proteins.