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     

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.     

Mass production of human epidermal growth factor using fed-batch cultures of recombinant Escherichia coli

Fed-batch cultures of recombinant E. coli HB101 harboring expression plasmid pTRLBT1 or pTREBT1, with acetate concentration monitoring, are investigated to obtain high cell density and large amounts of human epidermal growth factor (hEGF). The expression plasmid pTRlBT1 contains a synthetic hEGF gene attached downstream of the N-terminal fragment of the trp L gene preceded by the trp promoter. The expression plasmid pTREBT1 contains the same coding sequence attached downstream of the N-terminal fragment of the trp E gene preceded by the trp promoter, trp L gene, and attenuator region. E. coli harboring pTREBT1 produces 0.56 mg/L hEGE and immediately degrades it. On the other hand E. coli harboring pTRLBT1 produces 6.8 mg/L hEGF and does not decompose it. Prominent inclusion bodies are observed in E. coli cells harboring pTRLBT1 using an election microscope. To Cultivate E. coli harboring pTRLBT1, a fed-batch culture system, divided into a cell growth step and an hEGF production step, is carried out. The cells grow smoothly without acetate-induced inhibition. Cell concentration and hEGF quantity reach the high values of 21 g/L and 60 mg/L, respectively.     

Fed-batch cultivation ofEscherichia coli YK537 (pAET-8) for production ofphoA promoter-controlled human epidermal growth factor

Secretion of the expressed heterologous proteins can reduce the stress to the host cells and is beneficial to their recovery and purification. In this study, fed-batch cultures ofEscherichia coliYK537 (pAET-8) were conducted in a 5-L fermentor for the secretory production of human epidermal growth factor (hEGF) whose expression, was under the control of alkaline phosphatase promoter. The effects of feeding of glucose and complex nitrogen sources on hEGF production were investigated. When the fed-batch culture was conducted in a chemically defined medium, the cell density was 9.68 g/L and the secreted hEGF was 44.7 mg/L in a period of 60 h. When a complex medium was used and glucose was added in pH-stat mode, the secreted hEGF was improved to 345 mg/L. When the culture was fed with glucose at a constant specific rate of 0.25 gg⁻¹h⁻¹, hEGF reached 514 mg/L. The effects of adding a solution containing yeast extract and tryptone were further studied. Different rate of the nitrogen source feeding resulted in different levels of phosphate and acetic acid formation, thus affected hEGF expression. At the optimal feeding rate, hEGF production achieved 686 mg/L.     

A recombinant vaccine against hydatidosis: production of the antigen in Escherichia coli

A commercial process was developed for producing a recombinant vaccine against hydatidosis in farm animals. The vaccine antigen consisting of a surface protein of the oncospheres of the hydatid worm (Echinococcus granulosus), was produced as inclusion bodies in Escherichia coli. Fed-batch cultures of E. coli using Terrific broth in stirred bioreactors at 37°C, pH 7.0, and a dissolved oxygen level of 30% of air saturation produced the highest volumetric concentrations of the final solubilized antigen. An exponential feeding strategy proved distinctly superior to feeding based on pH-stat and DO-stat methods. The plasmid coding for the antigen was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at 4 h after initiation of the culture. The minimum IPTG concentration for full induction was 0.1 mM.     

Recombinant human epidermal growth factor inclusion body solubilization and refolding at large scale using expanded-bed adsorption chromatography from Escherichia coli

Amongst the various endogenous growth factors, epidermal growth factor (EGF) plays an important role in normal wound healing of tissue such as skin, cornea and gastrointestinal tract. Various studies have proved that supplementing recombinant human EGF (rhEGF) results in significant augmentation of wound healing. In the present work, a high level expression system with poly-arginine sequences was used for the production of recombinant human EGF (rhEGF) as inclusion bodies. The inclusion bodies were solubilized and the protein was refolded by using expanded-bed adsorption chromatography. The renatured protein was digested with appropriate concentration of trypsin and subsequently the digested rhEGF is purified by passing through ion-exchange chromatography (Toyopearl-SP) to obtain a biologically active protein. This process is the shortest process with reduced number of steps of purification, eliminates the usage of preparative reversed phase HPLC (RP-HPLC) for final purification, which is an expensive technique. The purified protein was analyzed by RP-HPLC, showing a purity >99% and size exclusion chromatography profile shows that there are minimal aggregates, with 99% renatured active protein. The purified rhEGF showed a specific activity of 5 × 10⁵ IU/mg protein, in comparison with NIBSC standard (1st International Standard of rDNA-derived EGF, Code 91/530). The process has been successfully adopted at 100 L fermentation scale and the rhEGF based formulation has been commercialized with brand name REGEN D, with excellent clinical results.     

The expression of insulin-like growth factor binding proteins in human hepatocellular carcinoma

Insulin-like growth factors (IGF), IGF receptors and IGF binding proteins (IGFBPs) play an important role in cell growth and differentiation. The liver is the major source of IGF-1 and at least two IGFBPs (IGFBP-1 and IGFBP-3). IGFBPs most often serve to attenuate the effects of IGF at the receptor level and thereby limit IGF-induced cell growth and differentiation. Although changes in IGFBP expression have been described during controlled liver growth such as hepatic regeneration following partial hepatectomy, there is limited knowledge of IGFBPs gene expression in uncontrolled growth or hepatocellular carcinoma. In the present study, we employed Northern blotting techniques to document the expression of IGFBP-1, 3 and 4 in normal human livers, cirrhotic and hepatocellular carcinoma tissues. The results revealed no differences in IGFBP-1, 3 and 4 mRNA levels between normal and cirrhotic tissues. However, the expression of all three IGFBPs mRNA were significantly down regulated in hepatocellular carcinoma tissues. These findings are in keeping with IGFBPs playing an important inhibitory role in the development and/or growth of hepatocellular carcinoma in humans.     

Expression, refolding, and characterization of a novel recombinant dual human stem cell factor

A novel recombinant dual human stem cell factor (rdhSCF) gene which consisted of a full-length hSCF(1-165aa) cDNA and a truncated hSCF (1-145aa) cDNA, linked by a peptide (GGGGSGGGGSGG) coding region, was constructed and cloned into Escherichia coli expression vector pET-22b. The rdhSCF was expressed at high level in E. coli BL21(DE3) and existed mainly as inclusion bodies. The inclusion bodies were solubilized in urea and refolded by ion-exchange chromatography. After renaturation, the purity of the yielded rdhSCF was up to 90%. Cell proliferation assay showed that the specific activity of the rdhSCF was 2.86x10(5) U/mg, about 1.66 times as high as that of monomer rhSCF expressed in E. coli.     

The production of recombinant beta-galactosidase inEscherichia coli in yeast extract enriched medium

Summary The productivity ofEscherichia coli biomass and recombinant beta-galactosidase was increased in Luria broth (LB) enriched with yeast extract. In flask culture under conditions of LB limitation, yeast extract suplementation gave the highest biomass (strain HB101/pRW756) stimulation per unit of component added compared with supplementation by various amounts of amino acids, vitamins, minerals, purines/pyrimidines, tryptone, casamino acids, casein peptone or gelatin peptone. The biomass production ofE. coli HB101/pRW756, XL-1 blue/puc118, XL-1 Blue FF/puc118 and TB-1/p1034 cells was stimulated in fermentor-scale experiments with additional yeast extract in LB. Total beta-galactosidase production from plasmid genes in fermentor-scale experiments was increased 105.4% in XL-1 blue/puc118 cells, 365.5% in XL-1 blue FF/puc118 cells and 421.4% in TB-1/p1034 cells by 0.5%, 1% and 1% weight per volume of additional yeast extract in LB, respectively. Depending on different strains, the increase of the enzyme production was obtained either by increased biomass, or the combination of enhanced gene expression and increased biomass. Neither the biomass nor beta-galactosidase production was stimulated in N4830/p1034 cells by the increase in yeast extract concentration in the medium.     

Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose

5-Aminolevulinic acid (ALA) recently received much attention due to its potential applications in many fields. In this study, we developed a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C5 pathway. The expression of a mutated hemA gene, encoding a glutamyl-tRNA reductase from Salmonella arizona, significantly improved ALA production from 31.1 to 176 mg/L. Glutamate-1-semialdehyde aminotransferase from E. coli was found to have a synergistic effect with HemA^M from S. arizona on ALA production (2052 mg/L). In addition, we identified a threonine/homoserine exporter in E. coli, encoded by rhtA gene, which exported ALA due to its broad substrate specificity. The constructed E. coli DALA produced 4.13 g/L ALA in modified minimal medium from glucose without adding any other co-substrate or inhibitor. This strategy offered an attractive potential to metabolic production of ALA in E. coli.     

Ethanol production from wheat straw by recombinant Escherichia coli strain FBR5 at high solid loading

Ethanol production by a recombinant bacterium from wheat straw (WS) at high solid loading by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid pretreated WS (150 g/L) after enzymatic saccharification was 86.3 ± 1.5 g/L. The pretreated WS was bio-abated by growing a fungal strain aerobically in the liquid portion for 16 h. The recombinant Escherichia coli strain FBR5 produced 41.1 ± 1.1 g ethanol/L from non-abated WS hydrolyzate (total sugars, 86.6 ± 0.3 g/L) in 168 h at pH 7.0 and 35 °C. The bacterium produced 41.8 ± 0.0 g ethanol/L in 120 h from the bioabated WS by SHF. It produced 41.6 ± 0.7 g ethanol/L in 120 h from bioabated WS by fed-batch SSF. This is the first report of the production of above 4% ethanol from a lignocellulosic hydrolyzate by the recombinant bacterium.     

Genetic engineering of Escherichia coli for production of tetrahydrobiopterin

Tetrahydrobiopterin (BH4) is an essential cofactor for various enzymes in mammals. In vivo, it is synthesized from GTP via the three-step pathway of GTP cyclohydrolase I (GCHI), 6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase (SPR). BH4 is a medicine used to treat atypical hyperphenylalaninemia. It is currently synthesized by chemical means, which consists of many steps, and requires costly materials and complicated procedures. To explore an alternative microbial method for BH4 production, we utilized recombinant DNA technology to construct recombinant Escherichia coli (E. coli) strains carrying genes expressing GCHI, PTPS and SPR enzymes. These strains successfully produced BH4, which was detected as dihydrobiopterin and biopterin, oxidation products of BH4. In order to increase BH4 productivity we made further improvements. First, to increase the de novo GTP supply, an 8-azaguanine resistant mutant was isolated and an additional guaBA operon was introduced. Second, to augment the activity of GCHI, the folE gene from E. coli was replaced by the mtrA gene from Bacillus subtilis. These modifications provided us with a strain showing significantly higher productivity, up to 4.0 g of biopterin/L of culture broth. The results suggest the possibility of commercial BH4 production by our method.     

Using a kinetic model that considers cell segregation to optimize hEGF expression in fed-batch cultures of recombinant Escherichia coli

Growth inhibition of recombinant Escherichia coli during the expression of human epidermal growth factor was observed. The recombinant cells could be segregated into three populations based on their cell division and plasmid maintenance abilities: dividing and plasmid-bearing cells, dividing and plasmid-free cells, and viable-but-non-culturable (VBNC) cells. Fed-batch fermentations were performed to investigate the effect of cell segregation on the kinetics of growth and foreign protein production. The results showed that a low concentration of inducer caused weak induction, whereas high levels cause strong induction, resulting in cells segregating into VBNC bacteria and producing a low foreign protein yield. A kinetic model for cell segregation was proposed and its predictions correlated well with experimental data for cell growth and protein expression. The optimal induction strategy could then be predicted by the model, and this prediction was then verified by experimentally deriving the conditions necessary for maximum expression of recombinant protein.     

Extracellular production of active vibriolysin engineered by random mutagenesis in Escherichia coli

Vibriolysin, an extracellular protease of Vibrio proteolyticus, is synthesized as a preproenzyme. The N-terminal propeptide functions as an intramolecular chaperone and an inhibitor of the mature enzyme. Extracellular production of recombinant vibriolysin has been achieved in Bacillus subtilis, but not in Escherichia coli, which is widely used as a host for the production of recombinant proteins. Vibriolysin is expressed as an inactive form in E. coli possibly due to the inhibitory effect of the N-terminal propeptide. In this study, we isolated the novel vibriolysin engineered by in vivo random mutagenesis, which is expressed as active mature vibriolysin in E. coli. The Western blot analysis showed that the N-terminal propeptide of the engineered enzyme was processed and degraded, confirming that the propeptide inhibits the mature enzyme. Two mutations located within the engineered vibriolysin resulted in the substitution of stop codon for Trp at position 11 in the signal peptide and of Val for Ala at position 183 in the N-terminal propeptide (where position 1 is defined as the first methionine). It was found that the individual mutations are related to the enzyme activity. The novel vibriolysin was extracellularly overproduced in BL21(DE3) and purified from the culture supernatant by ion-exchange chromatography followed by hydrophobic-interaction chromatography, resulting in an overall yield of 2.2 mg/L of purified protein. This suggests that the novel engineered vibriolysin is useful for overproduction in an E. coli expression system.     

Enhanced production of penicillin G acylase from a recombinant Escherichia coli

Penicillin G acylase (pac) gene was cloned into a stable asd ⁺ vector (pYA292) and expressed in Escherichia coli. This recombinant strain produced 1000 units penicillin G acylase g^–1 cell dry wt, which is 23-fold more than that produced by parental Escherichia coli ATCC11105. This enzyme was purified to 16 units mg^–1 protein by a novel two-step process.     

Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization

Thermolysin is a representative zinc metalloproteinase derived from Bacillus thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. Extracellular production of thermolysin has been achieved in Bacillus, but not in Escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. In this study, we expressed thermolysin as a single polypeptide pre-proenzyme in E. coli under the original promoter sequences in the npr gene, the gene from B. thermoproteolyticus, which encodes thermolysin. Active mature thermolysin (34.6 kDa) was secreted into the culture medium. The recombinant thermolysin was purified to homogeneity by sequential column chromatography procedures of the supernatant with hydrophobic-interaction chromatography followed by affinity chromatography. The purified recombinant product is indistinguishable from natural thermolysin from B. thermoproteolyticus as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-asparatyl-L-phenylalanine methyl ester. The results demonstrate that our expression system should be useful for structural and functional analysis of thermolysin.     

Changes of Escherichia coli cell cycle parameters during fast growth and throughout growth with limiting amounts of thymine

It is generally accepted that during fast growth of Escherichia coli, the time (D) between the end of a round of DNA replication and cell division is constant. This concept is not consistent with the fact that average cell mass of a culture is an exponential function of the growth rate, if it is also accepted that average cell mass per origin of DNA replication (M_i) changes with growth rate and negative exponential cell age distribution is taken into account. Data obtained from cell composition analysis of E. coli OV-2 have shown that not only (M_i) but also D varied with growth rate at generation times () between 54 and 30 min. E. coli OV-2 is a thymine auxotroph in which the replication time (C) can be lengthened, without inducing changes in , by growth with limiting amounts of thymine. This property has been used to study the relationship between cell size and division from cell composition measurements during growth with different amounts of thymine. When C increased, average cell mass at the end of a round of DNA replication also increased while D decreased, but only the time lapse (d) between the end of a replication round and cell constriction initiation appeared to be affected because the constriction period remained fairly constant. We propose that the rate at which cells proceed to constriction initiation from the end of replication is regulated by cell mass at this event, big cells having shorter d times than small cells.Abbreviations OD₄₅₀ and OD₆₃₀ Optical density at a given wavelength in nm Dedicated to Dr. John Ingraham to honor him for his many contributions to Science     

Optimization of BLyS production and purification from Escherichia coli

B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor superfamily of cytokines. When the 152 amino acids of the C-terminus are associated into a homotrimer, this protein exhibits the ability to stimulate B cell proliferation and differentiation. Since numerous potential therapeutic indications have been identified for BLyS and other BLyS-derived products, large quantities of the protein are needed to further basic research and clinical trials. In this work, we have developed a high yield recombinant expression system that utilizes Escherichia coli as the host organism. Recombinant soluble BLyS (rsBLyS) production was achieved through the use of the phoA promoter system. This expression system, coupled to a semi-defined fermentation process, resulted in final purified yields of 435 mg/L of properly folded, trimeric, biologically active rsBLyS. This level of production is an 11-fold increase in volumetric yields compared to the process currently being used for clinical production. Furthermore, the increased rsBLyS production obtained from this process enabled the development of a conventional purification scheme that eliminated the use of a BLyS-affinity resin.     

Efficient production by Escherichia coli of recombinant protein using salting-out effect protecting against proteolytic degradation

To produce glucoamylase efficiently as a recombinant protein, E. coli was grown with 20 g (NH₄)₂SO₄ l^–1 which removed proteolytic activity but did not effect cell growth. Growth in M9 medium with 20 g (NH₄)₂SO₄ l^–1 produced 11 U glucoamylase ml^–1 compared to 7 U ml^–1 without addition. Furthermore, the glucoamylase activity was maintained at about 9 U ml^–1.     

重组人表皮生长因子的稳定性

考察了不同条件下重组人表皮生长因子（rhEGF）的稳定性。结果表明,不同的保存条件（物理状态、温度、pH、浓度等）对rhEGF的稳定性有显影响,使用适当的添加剂（保护剂、抗氧剂、抑菌等）可以提高rhEGF的稳定性。上述结果为rhEGF的制剂学研究提供了依据。