Expression of secreted insulin-like growth factor-1 in Escherichia coli

The synthesis, processing and secretion of insulin-like growth factor-1 (IGF-1 or somatomedin-C) fused to LamB and OmpF secretion leader sequences in Escherichia coli have been investigated. Expression and secretion of IGF-1 was achieved. The major portion of this secreted IGF-1 accumulated in the periplasmic space as insoluble aggregates. A small amount of IGF-1 was found folded in its native conformation in the medium. The lamB and ompF signal sequences were fused to the 5' coding sequence of IGF-1. Fusion of the lamB signal sequence directly to IGF-1 (lamB-IGF-1) resulted in accumulation of 16-20 micrograms/A550/ml of correctly processed IGF-1 in the periplasmic space. The processing efficiency of LamB-IGF-1 and OmpF-IGF-1 was enhanced in an E. coli strain bearing a prlA4 mutation. Amino acid sequence analysis of IGF-1 secreted into the periplasm and exported into the medium confirmed the precise removal of the LamB or OmpF signal sequence. IGF-1 synthesized in E. coli was demonstrated to be active in a cell proliferation bioassay.     

Production of recombinant human granulocyte-colony-stimulating factor in high cell density yeast cultures

The recombinant human granulocyte-colony-stimulating factor (rhG-CSF) was synthesized in a fusion protein using a GAL1-10 UAS in recombinant Saccharomyces cerevisiae and the intracellular KEX2 cleavage led excretion of mature rhG-CSF into the extracellular culture broth. The recombinant yeast growth in fed-batch cultures was controlled by precise computer-aided medium feed. The optimal C/N ratio in preinduction (glucose/Casamino acids) and post-induction (galactose/yeast extract) feed media was determined at 3 and 2, respectively. The final rhG-CSF and cell concentration was more than 60 mg/L and 70 g/L, respectively, with around 90% plasmid stability and negligible ethanol accumulation. Comparing the cell growth between the hG-CSF + and hG-CSF - recombinant strains shows that the cloned gene product does not hamper the host cell growth.     

Recombinant trypsin production in high cell density fed-batch cultures in Escherichia coli

Fed-batch techniques were employed to obtain high cell density cultures (92-100 g DCW/L) of Escherichia coli strain X90 producing a recombinant serine protease, rat anionic trypsin, secreted to the periplasm. The specific growth rate was controlled to minimize growth-inhibiting acetate formation by utilizing an exponential feeding profile determined from mass balance equation. The volumetric yield of recombinant rat anionic trypsin was 56 mg/L, and the final cell density was 92 g DCW/L when the culture was induced in the late logarithmic phase. However, when the culture was induced in the early logarithmic phase, the volumetric yield was 13 mg/L and the final cell density was 14 g DCW/L. Thus, the induction timing is shown to have a significant effect on the final cell density as well as the overall volumetric yield of the recombinant protease. (c) 1993 Wiley & Sons, Inc.     

Production of thermally induced recombinant proteins relative to cell biomass is influenced by cell density in Escherichia coli batch cultures

Summary Fed-batch cultures of recombinant Escherichia coli were studied regarding their ability to permit CI857^ts-controlled gene expression at different growth phases. Cells, growing at 50% pO₂ and 28°C in a bioreactor, were transferred to a shaker flask at 42°C and the product yield was analysed after a short incubation time. Within the batch period, the synthesis of recombinant -galactosidase relative to the increase of biomass during the induction period was enhanced in an biomass-dependent fashion. Whereas in young cells biomass production dominates over recombinant protein production, the available energetic resources of aged cells seem to be employed more efficiently for CI857-controlled biosynthesis of recombinant proteins than of cell material. A model to describe this altruistic behaviour of old cultures is here proposed.     

Over-expression of recombinant human interferon-gamma in high cell density fermentation of Escherichia coli

Human interferon-gamma (hIFN-gamma) was expressed in Escherichia coli BL21(DE3) under the control of the T7 promoter. Glucose was used as the sole source of carbon and energy with simple exponential feeding rate in fed-batch process. Cell density of recombinant E. coli was reached to 100 g dry wt l(-1) under both constant (0.12 h(-1)) and variable (0.12-0.52 h(-1)) specific growth rates. In the variable specific growth rate fed-batch process, plasmid stability and specific yield of rhIFN-gamma were greater than constant specific growth rate fed-batch process. The final specific yield and overall productivity of rhIFN-gamma were 0.35 +/- 0.02 g rhIFN-gamma g(-1) dry cell wt and 0.9 +/- 0.05 g rhIFN-gamma l(-1) h(-1) in the variable specific growth rate fed-batch process, respectively.     

Production of bovine insulin-like growth factor 2 (bIGF2) in Escherichia coli

Bovine insulin-like growth factor 2 (bIGF2) was produced in inclusion bodies in the cytoplasm of Escherichia coli and accumulated at high levels: 20-25% of total Coomassie-stained bacterial protein. The level of accumulation of bIGF2 was affected by the choice of codons in the 5' end of the coding sequence and by a rpoH mutation in the host cells. Purified recombinant bIGF2 had the native N terminus and the same mitogenic activity as that of bIGF2 purified from bovine serum.     

Temperature-induced production of recombinant human insulin in high-cell density cultures of recombinant Escherichia coli

The construction of expression vectors encoding either the human insulin A- or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported. Using this two-chain approach we also describe the separate isolation of the insulin A- and B-chains from inclusion bodies and their subsequent assembly into native human insulin. The production of the insulin fusion proteins were carried out in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source. The expression of the recombinant genes by temperature-shift in high-cell density cultures of recombinant E. coli resulted in product yields of grams per litre of culture broth, e.g. 4.5 g of insulin B-chain fusion protein per litre of culture broth. This translates into an expression yield of about 800 mg of the insulin B-chain per litre of culture. Under similar cultivation conditions the expression yield of the insulin A-chain corresponds to approximately 600 mg per litre of culture. The metabolic burden imposed on the recombinant cells during temperature-induced production of insulin fusion proteins in high-cell density cultures is reflected in an increased respiratory activity and a reduction of the biomass yield coefficient with respect to glucose.     

Production of a platelet aggregation inhibitor, salmosin, by high cell density fermentation of recombinant Escherichia coli

Optimal conditions for a high cell density fermentation were investigated in a recombinant Escherichia coli producing salmosin, a platelet aggregation inhibitor. The optimized carbon and nitrogen sources were glycerol 10 g/l, yeast extract 30 g/l, and bacto-tryptone 10 g/l, yielding the dry cell weight (DCW) of 10.61 g/l in a 500 ml flask culture. The late-stage induction with 1% L-arabinose in a 5 l jar fermentor showed the highest DCW of 65.70 g/l after 27 h of the fed-batch fermentation. Around 2,200 mg/l of the protein was expressed as an inclusion body that was then refolded to obtain the active salmosin of 96 mg/l. We also confirmed the inhibitory activity against platelet aggregation of the active salmosin from the high cell density fermentation.     

Use of high density cultures of Escherichia coli for high level production of recombinant Pseudomonas aeruginosa exotoxin A

Summary An efficient fermentation method for the production of two modified recombinant Pseudomonas aeruginosa exotoxin As cloned in Escherichia coli BL21(DE3) was developed. Cell densities of 16–30 g dry weight/l were found to be most suitable for the induction of protein synthesis, which was under the isopropyl \-d-thiogalactopyranoside (IPTG)-inducible T7 expression system. A concentration of 0.6 mm IPTG and induction time of 90 min were found to give the best results for production of the modified toxins. Using this procedure, gram amounts of the proteins were obtained in a 3-1 bench-top fermentor. The high density growth of the bacteria did not impair the integrity of the proteins and did not interfere with the purification procedure. Offsprint requests to: J. Shiloach     

Process development for production of recombinant human insulin-like growth factor-I in Escherichia coli

Fed-batch cultures were carried out to overproduce human insulin-like growth factor I (IGF-I) in Escherichia coli. The effects of carbon sources (glucose or glycerol) and induction time on cell growth and IGF-I production were investigated in more detail. Glycerol was a better carbon source than glucose for IGF-I production in fed-batch culture. Induction at the mid-exponential phase with glycerol as a carbon source in the pH-stat fed-batch culture was optimal for IGF-I production. Under this condition, 2.8 g L⁻¹ of fusion IGF-I was produced as inclusion bodies. We have also developed downstream processing for preparative scale purification of IGF-I from the fusion protein produced by the fed-batch culture using glycerol as a carbon source. After the fusion protein expressed was solubilized in 8 M urea and cleaved with hydroxylamine, the released IGF-I was purified by cation exchange chromatography, refolding and preparative scale reverse phase HPLC (rp-HPLC) to give recombinant IGF-I of >98% purity. The biological activities of the purified IGF-I were measured and found to be identical to those of commercial IGF-I. Journal of Industrial Microbiology & Biotechnology (2000) 24, 94–99. Received 13 January 1999/ Accepted in revised form 02 October 1999     

Fuzzy neural network for the control of high cell density cultivation of recombinant Escherichia coli

A five-layer fuzzy neural network (FNN) was developed for the control of fed-batch cultivation of recombinant Escherichia coli JM103 harboring plasmid pUR 2921. The FNN was believed to represent the membership functions of the fuzzy subsets and to implement fuzzy inference using previous experimental data. This FNN was then used for compensating the exponential feeding rate determined by the feedforward control element. The control system is therefore a feedforward-feedback type. The change in pH of the culture broth and the specific growth rate were used as the inputs to FNN to calculate the glucose feeding rate. A cell density of 84 g DWC/l in the fed-batch cultivation of the recombinant E. coli was obtained with this control strategy. Two different FNNs were then employed before and after induction to enhance plasmid-encoded β-galactosidase production. Before induction the specific growth rate was set as 0.31 h⁻¹, while it was changed to 0.1 h⁻¹ after induction. Compared to when only one FNN was used, the residual glucose concentration could be tightly controlled at an appropriate level by employing two FNNs, resulting in an increase in relative activity of β-galactosidase which was about four times greater. The present investigation demonstrates that a feedforward-feedback control strategy with FNN is a promising control strategy for the control of high cell density cultivation and high expression of a target gene in fed-batch cultivation of a recombinant strain.     

Control of Escherichia coli growth rate through cell density

The transition from the exponential to the stationary phase of Escherichia coli cultures has been investigated regarding nutrient availability. This analysis strongly suggests that the declining of the cell division rate is not caused by mere nutrient limitation but also by an immediate sensing of cell concentration. In addition, both the growth rate and the final biomass achieved by a batch culture can be manipulated by altering its density during the early exponential phase. This result, which has been confirmed by using different experimental approaches, supports the hypothesis that the E. coli quorum sensing is not only determined by the release of soluble cell-to-cell communicators. Cell-associated sensing elements might also be involved in modulating the bacterial growth even in the presence of non-limiting (although declining) nutrient concentrations, thus promoting their economical utilisation in dense populations.     

Recombinant protein production in high cell density cultures of Escherichia coli with galactose as a gratuitous inducer

A new expression system was developed by introducing two major modifications into the genome of Escherichia coli: a deletion in the gal operon (DeltagalEKT) to allow the use of the inexpensive compound galactose as a gratuitous inducer and the introduction of the gal P2 promoter driving the expression of the T7 RNA polymerase. The novel JRR10 strain containing these two features gives high-level expression of a reporter gene cloned under the T7 phi10 promoter in high cell density cultures. The cost of the induction of this novel system is more than 30 times lower than that of the IPTG-induced system of the widely used BL21 strain.     

Production of recombinant serpins in Escherichia coli

Expression systems based on Escherichia coli offer fast, cheap, and convenient means for the production of recombinant serpins. Over 30 active serpins from prokaryotic and eukaryotic organisms have been produced in this way, using a variety of vectors, promoters, fusion partners, and host strains. Serpins forming insoluble inclusion bodies in E. coli can generally be solubilized and refolded. Here, we outline the general approaches and procedures to be considered when contemplating the use of E. coli for recombinant serpin production.     

High cell density and high expression of recombinant human ApoA-IMilano in Escherichia coli by twice temperature-shifted induction

The effect of temperature on the formation of recombinant protein, apolipoprotein A-I_Milano was investigated in the present study. The temperature of the initial growth phase was set at 30°C, while temperature variation in induction phase was arranged in three modes. High cell-density culture of Escherichia coli and high expression of recombinant human by twice temperature-shifted induction were carried out. Experimental results showed that ApoA-I_Milano reached 4.8 g/L with the final cell density of OD₆₀₀, 150. It was found that twice temperature-shifted induction could successfully avoid the effect of acetic acid on cell density and the expression of the product. The present study provides a basic procedure for the production of recombinant ApoA-I_Milano.     

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.     

Cultivation of recombinant Escherichia coli to achieve high cell density with a high level of penicillin G acylase activity

A mutant strain of E. coli EP1 harbouring pGL-5 was employed to develop a process for producing penicillin G acylase (PGA). In comparison with different carbon sources in the medium, it was found that the specific levels of PGA activity obtained in the glucose medium were the lowest. which was likely due to catabolic repression. Phenylacetic acid (PAA) was previously reported to be an regulatory inducer for PGA production, whereas in this study, the addition of PAA repressed both cell growth and enzyme expression. In a fed-batch culture, the increase of specific PGA activity followed the pattern of the cell concentration during the early to middle cell growth phase. With application of pure oxygen aeration and an appropriate medium design, the cell concentration reached 162 (g wet weight/l), which was 2.4 times higher compared to that of the original operation, and a specific PGA activity of 37 (IU/g wet weight) was achieved after 12 h of cultivation.     

Production of recombinant Chikungunya virus envelope 2 protein in Escherichia coli

Chikungunya, a mosquito-borne viral disease caused by Chikungunya virus (CHIKV), has drawn substantial attention after its reemergence causing massive outbreaks in tropical regions of Asia and Africa. The recombinant envelope 2 (rE2) protein of CHIKV is a potential diagnostic as well as vaccine candidate. Development of cost-effective cultivation media and appropriate culture conditions are generally favorable for large-scale production of recombinant proteins in Escherichia coli. The effects of medium composition and cultivation conditions on the production of recombinant Chikungunya virus E2 (rCHIKV E2) protein were investigated in shake flask culture as well as batch cultivation of Escherichia coli. Further, the fed-batch process was also carried out for high cell density cultivation of E. coli expressing rE2 protein. Expression of rCHIKV E2 protein in E. coli was induced with 1 mM isopropyl-beta-thiogalactoside (IPTG) at ~23 g dry cell weight (DCW) per liter of culture and yielded an insoluble protein aggregating to form inclusion bodies. The final DCW after fed-batch cultivation was ~35 g/l. The inclusion bodies were isolated, solubilized in 8 M urea and purified through affinity chromatography to give a final product yield of ~190 mg/l. The reactivity of purified E2 protein was confirmed by Western blotting and enzyme-linked immunosorbent assay. These results show that rE2 protein of CHIKV may be used as a diagnostic reagent or for further prophylactic studies. This approach of producing rE2 protein in E. coli with high yield may also offer a promising method for production of other viral recombinant proteins.     

Direct chemical extraction of a recombinant viral coat protein from Escherichia coli at high cell density.

The release of protein and DNA from nonrecombinant E. coli JM101 and recombinant E. coli HMS174(DE3) expressing L1 (the major viral coat protein of human papillomavirus type 16) as an inclusion body was demonstrated at high cell density (OD(600) = 160). For the nonrecombinant strain, extraction efficiency decreased significantly as cell mass increased, with a high viscosity increase in the postextraction broth. A different dependence on cell concentration was observed for the recombinant strain, with total protein extraction efficiency exceeding 85% for both uninduced and induced cells. Almost complete release of the recombinant L1 protein was achieved at high cell concentration (OD(600) = 80 approximately 160) without the use of reducing agent. This greatly extends the concentration range for chemical extraction.