Effects of induction starting time and Ca2+ on expression of active penicillin G acylase in Escherichia coli

Formation of inclusion bodies is an important obstacle to the production of active recombinant protein in Escherichia coli. Thus, soluble expression of penicillin G acylase from Kluyvera citrophila was investigated in BL21(DE3). In this study, the yield of active enzyme was significantly enhanced by the composition of the medium and induction opportunity. When 0.5 mmol/L IPTG was added to complex medium at 15 h after incubation, the volumetric and specific activities of penicillin G acylase both achieved the highest values, respectively. However, aggravation of intracellular proteolysis and decline of enzyme expression were also observed if induction occurred too much later. Ca2+ ion was another critical factor in cell growth and protein expression. When 24 mmol/L Ca2+ ion was adding to the medium at the beginning of fermentation, a greater than 2-fold increase in cell density and a 7-fold increase in volumetric activity of penicillin G acylase were reached. Nevertheless, no significant benefit for recombination protein expression was found when excess Ca2+ was added after induction time. This study demonstrates that the induction starting time and Ca2+ ion are two critical factors for the expression of active penicillin G acylase.     

Optimization of fusion proinsulin production by high cell-density fermentation of recombinantE. coli

The optimum conditions for mass production of fusion proinsulin were studied in recombinantEscherichia coli strain BL21 (DE3) [pT7-PI] using fed-batch culture employing pH-stat method. Yeast extract was found to enhance both the growth rate of recombinantE. coli strain BL21 (DE3) [pT7-PI] and its cell mass yield. When the glucose concentration was 10 g/L in the initial medium, 10 g/L concentration of yeast extract was found to be optimal to control the acetate production and to augment both the cell mass yield and the growth rate. Optimum ratio of glucose to yeast extract to minimize the cost of the feeding medium in the fed-batch culture was calculated to be 1.225 and verified by the subsequent experiments. The appropriate inducer concentration and induction time were examined with isopropyl-β-D-thiogalactopyranoside (IPTG). Irrespective of the induction time, IPTG induction resulted in the reduction of growth rate, but the expression level of the fusion protein was maintained at the level of about 20% of the total proteins. Since the volumetric productivity was well maintained in the range between 0.15 and 0.18 g/L.hr at the inducer concentration of above 0.025 mM, the appropriate inducer concentration, in relation to the inducer cost, is considered to be about 0.025 mM.     

AN OPTIMIZED PROTOCOL FOR OVERPRODUCTION OF RECOMBINANT PROTEIN EXPRESSION IN Escherichia coli

The gram-negative bacterium Escherichia coli (E. coli) offers a means for rapid, high-yield, and economical production of recombinant proteins. Here, a protocol for optimization of parameters involved in bacterial expression conditions is described. L-Asparaginase (ASNase II) was chosen as a model protein for our experiments. ASNase II gene (ansB) was cloned into the pAED4 plasmid and transformed into E. coli BL21pLysS (DE3)-competent cells. It was assumed that high cell density and high copy number of recombinant plasmid in the bacteria host could result in very high production of the recombinant protein. Circumstances for the overproduction of recombinant ASNase II including cell growth conditions, isopropyl β-D-1-thiogalactopyranoside (IPTG) level, ampicillin (Amp) concentration before and during IPTG induction, and cell density were optimized. Regarding the final optimization, overexpression of ASNase II was assessed on a large scale in LB medium. Periplasmic ASNase II was extracted using an alkaline lysis method. The extracted protein was purified by one-step DEAE-Sepharose fast-flow chromatography. ASNase II activity was considered an index for the protein expression. Applying the optimized practical protocol, protein production was significantly enhanced in comparison to the traditional IPTG induction method in the absence of a fermentor and can be applied for overexpression of other recombinant proteins.     

Reduction of acetate accumulation in Escherichia coli cultures for increased recombinant protein production

The culture of Escherichia coli for the commercial production of recombinant proteins has increased significantly in recent years. The production of acetate as a byproduct retards cell growth, inhibits protein formation, and diverts carbon from biomass to protein product. Our approach to reducing acetate accumulation was to disable the phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) by deleting the ptsHI operon in the wild-type E. coli strain GJT001. The mutation caused a severe reduction in growth rate and glucose uptake rate in glucose-supplemented M9 minimal medium, which confirmed the mutation, and eliminated acetate accumulation. The mutant strain (TC110) apparently metabolized glucose by a non-PTS mechanism that we are currently investigating, followed by phosphorylation by glucokinase. In complex medium such as 2xLB broth with 2% glucose, TC110 was able to grow quickly and still retained the phenotype of significantly reduced acetate accumulation (9.1+/-6.6 vs. 90.4+/-1.6mM in GJT001, P<0.05). The reduced acetate accumulation resulted in a significant improvement in final OD (23.5+/-0.7 in TC110 vs. 8.0+/-0.1 in GJT001, P<0.05). We tested the strains for the production of model recombinant proteins such as green fluorescent protein (GFP) and beta-galactosidase. TC110 had a 385-fold improvement in final volumetric productivity of GFP over GJT001 in shake flasks with 2xLB broth with 2% glucose. The distribution of GFP fluorescence in the cell population, as determined by flow cytometry, was much broader in GJT001 (coefficient of variation=466+/-35%) than in TC110 (coefficient of variation=55+/-1%). In corn steep liquor medium with 2% glucose, we observed a 28.5-fold improvement in final volumetric production of GFP in TC110 over GJT001. TC110 had a 7.5-fold improvement in final volumetric productivity of beta-galactosidase over GJT001 in 2xLB broth with 2% glucose medium. When tested in a batch bioreactor cultures with 2xLB broth with 2% glucose medium, the volumetric production of GFP by TC110 was 25-fold higher than that of GJT001. In summary, the ptsHI mutant of GJT001 resulted in reduced acetate accumulation, which led to significant improvements in recombinant protein production in batch bioreactors.     

Pseudomonas aeruginosa amidase: aggregation in recombinant Escherichia coli

The effect of cultivation parameters such as temperature incubation, IPTG induction and ethanol shock on the production of Pseudomonas aeruginosa amidase (E.C.3.5.1.4) in a recombinant Escherichia coli strain in LB ampicillin culture medium was investigated. The highest yield of soluble amidase, relatively to other proteins, was obtained in the condition at 37°C using 0.40 mM IPTG to induce growth, with ethanol. Our results demonstrate the formation of insoluble aggregates containing amidase, which was biologically active, in all the tested growth conditions. Addition of ethanol at 25°C in the culture medium improved amidase yield, which quantitatively aggregated in a biological active form and exhibited in all conditions an increased specific activity relatively to the soluble form of the enzyme. Non-denaturing solubilization of the aggregated amidase was successfully achieved using L-arginine. The aggregates obtained from conditions at 37°C by FTIR analysis demonstrated a lower content of intermolecular interactions which facilitated the solubilization step applying non-denaturing conditions. The higher interactions exhibited in aggregates obtained at suboptimal conditions compromised the solubilization yield. This work provides an approach for the characterization and solubilization of novel reported biologically active aggregates of this amidase.     

IPTG limitation avoids metabolic burden and acetic acid accumulation in induced fed-batch cultures of Escherichia coli M15 under glucose limiting conditions

The most common strategy to produce recombinant proteins using Escherichia coli as expression vector is fed-batch culture, since high cell density cultures strategies have successfully been applied. Several methodologies to limit the specific growth rate in order to control E. coli metabolism have been defined, demonstrating that cultures can be grown under glucose limitation up to high cell densities without accumulation of acetic acid. However, under induction conditions it has been observed that E. coli metabolism is reorganized again and leads to acetic acid accumulation, causing inhibition of cell growth and decreasing protein expression efficiency.We propose a double limitation strategy (glucose and IPTG) for E. coli fed-batch cultures to avoid the deregulation of the metabolism in the induction phase. Reducing the concentration of IPTG while keeping glucose growth limitation, the accumulation of acetic acid decreased. At an IPTG concentration of 0.03 mmol/g DCW no accumulation of acetic acid was observed during the induction phase, in contraposition to what has normally been observed.Although a slight reduction of protein expression rate was observed when applying this double limitation strategy, the bioprocess volumetric productivity was enhanced due to the capability to prolong the induction phase, reaching higher levels of protein production. Another advantage of this strategy is the reduction of media cost due to the lower level of IPTG used.     

Production of a secreted glycoprotein from an inducible promoter system in a perfusion bioreactor

The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated.     

Characterization of a pH-inducible promoter system for high-level expression of recombinant proteins in Escherichia coli

A pH-inducible promoter system was characterized and its potential applicability in recombinant protein production was evaluated using a plasmid construct, pSM552-545C(-), in which the promoter and activator coding sequences of the cad operon were inserted into the upstream region of a lacZ' reporter gene. Graded gene expression levels with respect to culture pH between 8.0 and 5.5 were observed and the induction range can be as high as 200-fold. The effects of several cultivation parameters, including pH, temperature, induction cell density, and inoculum size, were systematically examined. The practical application of this expression system to high level production of recombinant proteins was successfully demonstrated using a rich medium, superbroth. An extremely high recombinant protein productivity at a value of approximately 1.4 g/L with a specific expression level as high as 35% of total cellular protein can be obtained in a simple batch cultivation. The behavior of this expression system was further investigated using chemostat cultures. An uncommon relationship between the volumetric or specific recombinant protein activity and the dilution rate, with a maximal activity at a dilution rate of approximately 0.4 h(-1)was observed. (c) 1995 John Wiley & Sons, Inc.     

Factors influencing excretive production of human epidermal growth factor (hEGF) with recombinant Escherichia coli K12 system

Human epidermal growth factor (hEGF) was expressed and excreted into culture medium in an excretory recombinant Escherichia coli system. Conditions for the production of hEGF in this system were investigated. Results showed that the slight reduction of promoter strength improved the stability of plasmid and the production of hEGF in this recombinant system. One favorable MMBL medium for hEGF production was formulated by evaluating the effects of medium components, ampicilin addition and pH. hEGF production was affected obviously by culture conditions, especially fermentation temperature. High temperature (32 °C) was very beneficial for culture process by increasing productivity and reducing the quantity of isopropyl-#-d-thiogalactopyranoside (IPTG) for sufficient induction. High cell density and hEGF productivity could be accomplished concomitantly by inducing the culture at middle or late log phase of cell growth. In comparison with the batch process, fed-batch cultivation could improve plasmid stability from ca. 72% to ca. 83%, and increased hEGF productivity by 24.4%. Under all these circumstances, almost all expressed hEGF (⃋%) was fully excreted into the culture medium.     

Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli

Aim:  To investigate the effects of feeding and induction strategies on the production of Bm R1 recombinant antigen.
Methods and Results:  Fed-batch fermentation was studied with respect to the specific growth rate and mode of induction to assess the growth potential of the bacteria in a bioreactor and to produce high yield of Bm R1 recombinant antigen. Cells were grown at a controlled specific growth rate (μ_set) during pre-induction, followed by constant feeding postinduction. The highest biomass (24·3 g l⁻¹) was obtained during fed-batch process operated at μ_set of 0·15 h⁻¹, whereby lower μ_set (0·075 h⁻¹) gave the highest protein production (9·82 mg l⁻¹). The yield of Bm R1 was increased by 1·2-fold upon induction with 1 mmol l⁻¹ IPTG (isopropyl-β- d -thiogalactoside) compared to using 5 mmol l⁻¹ and showed a further 3·5-fold increase when the culture was induced twice at the late log phase.
Conclusions:  Combination of feeding at a lower μ_set and twice induction with 1 mmol l⁻¹ IPTG yielded the best result of all variables tested, promising an improved method for Bm R1 production .
Significance and Impact of the Study:  This method can be used to increase the production scale of the Bm R1 recombinant antigen to meet the increasing demand for Brugia Rapid^™, a commercial diagnostic test for detection of brugian filariasis.     

Evidencing the role of lactose permease in IPTG uptake by Escherichia coli in fed-batch high cell density cultures

The lac-operon and its components have been studied for decades and it is widely used as one of the common systems for recombinant protein production in Escherichia coli. However, the role of the lactose permease, encoded by the lacY gene, when using the gratuitous inducer IPTG for the overexpression of heterologous proteins, is still a matter of discussion. A lactose permease deficient strain was successfully constructed. Growing profiles and acetate production were compared with its parent strain at shake flask scale. Our results show that the lac-permease deficient strain grows slower than the parent in defined medium at shake flask scale, probably due to a downregulation of the phosphotransferase system (PTS). The distributions of IPTG in the medium and inside the cells, as well as recombinant protein production were measured by HPLC-MS and compared in substrate limiting fed-batch fermentations at different inducer concentrations. For the mutant strain, IPTG concentration in the medium depletes slower, reaching at the end of the culture higher concentration values compared with the parent strain. Final intracellular and medium concentrations of IPTG were similar for the mutant strain, while higher intracellular concentrations than in medium were found for the parent strain. Comparison of the distribution profiles of IPTG of both strains in fed-batch fermentations showed that lac-permease is crucially involved in IPTG uptake. In the absence of the transporter, apparently IPTG only diffuses, while in the presence of lac-permease, the inducer accumulates in the cytoplasm at higher rates emphasizing the significant contribution of the permease-mediated transport.     

Improvement of heterologous protein productivity using recombinant Yarrowia lipolytica and cyclic fed-batch process strategy

A cyclic fed-batch bioprocess is designed and a significant improvement of rice alpha-amylase productivity of recombinant Yarrowia lipolytica is illustrated. A bioprocess control strategy developed and reported here entails use of a genetically stable recombinant cloned for heterologous protein, use of optimized media for cell growth and enzyme production phases, and process control strategy enabling high cell-density culture and high alpha-amylase productivity. This process control can be achieved through maintaining a constant optimal specific cell growth rate at a predetermined value (i.e., 0.1 h-1), controlling medium feed rate commensurate with the cell growth rate, and maintaining a high cell-density culture (i.e., 60-70 g/L) for high productivity of cloned heterologous protein. The volumetric enzyme productivity (1, 960 units/L. h) achieved from the cyclic fed-batch process was about 3-fold higher than that of the fed-batch culture process (630 units/L. h).     

Batch and fed-batch cultivation for excretive production of human epidermal growth factor (hEGF) with recombinant E. Coli K12 system

Batch and fed-batch production of recombinant human epidermal growth factor (hEGF) was studied in an E. coli secretary expression system. By using MMBL medium containing 5 g/L glucose, controlling the temperature at 32 degrees C and maintaining the dissolved oxgen level over 20% saturation, a high yield of hEGF (32 mg/L) was obtained after an 18 hr batch cultivation with 0.2 mM IPTG induction at mid-log phase. Three different glucose feeding strategies were employed to further improve hEGF productivity in a bench top fermentor. Compared with the batch results, hEGF yield was improved up to 25.5% or 28.1%, respectively by intermittent or pH-stat glucose feeding, and up to 150% improvement of hEGF production was achieved by constant feeding of 200 g/L glucose solution at a rate of 0.11 mL/min. The effects of further combined feeding with other medium components and inducer on hEGF yield were also examined in the benchtop fermentor. This work is very helpful to further improve the productivity of extracellular hEGF in the recombinant E. coli system.     

Functional expression of single-chain variable fragment antibody against c-Met in the cytoplasm of Escherichia coli

c-Met, a high affinity receptor for hepatocyte growth factor/scatter factor, shown to be overexpressed in a variety of malignant cells, is a potential biomarker as well as a therapeutic target. Thus, single-chain antibody fragment (scFv) specific for c-Met is expected to be efficiently employed in the clinical treatment or imaging of many cancer cells. Here, we constructed the expression system for anti-c-Met scFv fused with T7 tag at its N-terminus using pET vector and investigated the expression conditions to achieve a functional and soluble expression of the scFv in the cytoplasm of recombinant Escherichia coli. The redox potential of E. coli cytoplasm was the most critical factor for the functional expression of anti-c-Met scFv. The employment of a host with oxidizing cytoplasm, E. coli trxB/gor double mutant, improved the productivity of functional anti-c-Met scFv by approximately 10-fold compared to the production of anti-c-Met scFv in the reducing cytoplasm of wild type E. coli. Productivity of functional anti-c-Met scFv could be further enhanced by co-expressing molecular chaperones such as GroELS, trigger factor, and DsbC with the scFv. Coexpression of DsbC increased the yield of functional anti-c-Met scFv about 2.5-fold in the cytoplasm of E. coli trxB/gor mutant compared to the production of scFv without DsbC coexpression. Lowering the IPTG concentration from 1 to 0.05 mM led to the slight enhancement, approximately 1.6-fold, of productivity of functional scFv. Although the use of low temperature for anti-c-Met scFv expression increased the ratio of soluble scFv fraction to insoluble fraction, productivity of soluble scFv decreased owing to the significant reduction of expression rate. The addition of 0.5 M sucrose in the medium inhibited the formation of intracellular insoluble anti-c-Met scFv. To purify the anti-c-Met scFv simply, we fused hexahistidine at the C-terminus of scFv and purified the scFv showing 98% of purity through the interaction between Ni2+ and histidine.     

High-level expression of soluble human β-defensin-2 in Escherichia coli

Human β-defensin-2 (hBD2) is a short cationic peptide with a broad antimicrobial spectrum. The coding sequence of hBD2 was cloned into pET-32a (+) to construct a fusion expression plasmid, pET32–hBD2, which was transformed into E. coli BL21 (DE3) for expression. The cultivation parameters of the expression vector harboring strain were optimized to produce the fusion protein in soluble form efficiently and to avoid the formation of insoluble inclusion bodies. The optimal conditions were determined as following: cultivation at 28 °C in MBL medium, induction at middle stage of exponential growth with 0.8 mM IPTG, and post-induction expression for 8 h. Under the above conditions, a high percentage of the target fusion protein (≥92.3%) was expressed in soluble form and the volumetric productivity of soluble fusion protein reached 1.3 g/l. The culture process was successfully scaled up in a 10 l bench-top fermentor.     

Kinetics of inclusion body production in batch and high cell density fed-batch culture of Escherichia coli expressing ovine growth hormone.

A process for maximizing the volumetric productivity of recombinant ovine growth hormone (r-oGH) expressed in Escherichia coli during high cell density fermentation process has been devised. Kinetics of r-oGH expression as inclusion bodies and its effect on specific growth rates of E. coli cells were monitored during batch fermentation process. It was observed that during r-oGH expression in E. coli, the specific growth rate of the culture became an intrinsic property of the cells which reduced in a programmed manner upon induction. Nutrient feeding during protein expression phase of the fed-batch process was designed according to the reduction in specific growth rate of the culture. By feeding yeast extract along with glucose during fed-batch operation, high cell growth with very little accumulation of acetic acid was observed. Use of yeast extract helped in maintaining high specific cellular protein yield which resulted in high volumetric productivity of r-oGH. In 16 h of fed-batch fermentation, 3.2 g l-1 of r-oGH were produced at a cell OD of 124. This is the highest concentration of r-oGH reported to date using E. coli expression system. The volumetric productivity of r-oGH was 0.2 g l-1 h-1, which is also the highest value reported for any therapeutic protein using IPTG inducible expression system in a single stage fed-batch process.     

Effect of re-feed strategies and non-ammoniagenic medium on adenovirus production at high cell densities

Recombinant adenoviruses became one of the vectors of choice for delivery and expression of foreign proteins for gene therapy and vaccination purposes. Nevertheless, the production of adenovirus is currently limited by the so-called "cell density effect", i.e., a drop in cell specific productivity concomitant with increased cell concentration at infection (CCI). This work describes the characterisation and optimisation of the infection process in order to improve recombinant adenovirus type 5 yields at high cell densities. For that purpose, 293 cells adapted to suspension were grown in 2l bioreactors and infected at different cell concentrations, using different re-feed strategies, while evaluating cell metabolism. The consumption of amino acids is enhanced during infection, although no amino acid limitation was detected for cells infected at concentrations in the range of 2 x 10(6)cell/ml, for which the highest volumetric productivity was obtained in batch mode. Conversely, infecting at cell concentrations in the range of 3 x10(6)cell/ml led to complete depletion of glucose, glutamine and threonine before the optimal harvesting time, a significant decrease in volumetric productivity being observed; the effect of amino acids and glucose addition at infection time on cell specific and volumetric productivity of adenovirus was assessed, no improvement on adenovirus production being achieved. The effect of ammonia, present in high concentrations at 3 x10(6)cell/ml, was evaluated and seem to be detrimental; an 1.8-fold increase on adenovirus volumetric productivity was obtained for infections performed at 3 x10(6)cell/ml when non-ammoniagenic medium was used.     

Enhancement of extracellular pullulanase production from recombinant Escherichia coli by combined strategy involving auto-induction and temperature control

Pullulanase was extracellularly produced with an engineered Escherichia coli with a combined strategy. When auto-induction instead of isopropyl β-d-1-thiogalactopyranoside (IPTG) induction method was implemented, we observed increased extracellular activity (4.2 U ml^?1) and cell biomass (7.95 g DCW l^?1). Subsequent investigation of temperature effect on fermentation showed cultivation performed at 25 °C presented the highest extracellular titer and cell biomass. In order to reduce the extended production period, we developed a two-stage temperature control strategy. Its application not only reduced the production period from 72 to 36 h, but also further enhanced the yield of extracellular pullulanase. Finally, with a view to releasing more intracellular pullulanase, we altered cell membrane permeability with various medium additives. As a result, extracellular titer was elevated to 68.23 U ml^?1, nearly 35-fold higher than that with IPTG induction method. The combined strategy developed here may be useful for the production of other extracellular proteins by recombinant E. coli.