Refolding and purification of recombinant human interferon-γ expressed as inclusion bodies inEscherichia coli using size exclusion chromatography

A size exclusion chromatography (SEC) process, in the presence of denaturant in the refolding buffer was developed to refold recombinant human interferon-γ (rhIFN-γ) at a high concentration. The rhIFN-γ was overexpressed inE. coli, resulting in the formation of inactive inclusion bodies (IBs). The IBs were first solubilized in 8 M urea as the denaturant, and then the refolding process performed by decreasing the urea concentration on the SEC column to suppress protein aggregation. The effects of the urea concentration, protein loading mode and column height during the refolding step were investigated. The combination of the bufferexchange effect of SEC and a moderate urea concentration in the refolding buffer resulted in an efficient route for producing correctly folded rhIFN-γ, with protein recovery of 67.1% and specific activity up to 1.2×10⁷ IU/mg.     

Over-production of human interferon-γ by HCDC of recombinant Escherichia coli

A simple fed-batch process was developed using a modified variable specific growth rate feeding strategy for high cell density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-gamma (hIFN-γ). The feeding rate was adjusted to achieve the maximum attainable specific growth rate during fed-batch cultivation. In this method, specific growth rate was changed from a maximum value of 0.55 h⁻¹ at the beginning of feeding and then it was reduced to 0.4 h⁻¹ at induction time.The final concentration of biomass and IFN-γ was reached to ∼115 g l⁻¹ (DCW) and 42.5 g(hIFN-γ) l⁻¹ after 16.5 h, also the final specific yield and overall productivity of recombinant hIFN-γ (rhIFN-γ) were obtained 0.37 g(hIFN-γ) g⁻¹ DCW and 2.57 g(hIFN-γ) l⁻¹ h⁻¹, respectively. According to available data this is the highest specific yield and productivity that has been reported for recombinant proteins production yet.     

Increased extracellular recovery of recombinant thermostable α-amylase from protein-excreting Escherichia coli cells by a simple water-wash procedure

Extracellular recovery of a recombinant, thermostable -amylase produced by Escherichia coli was increased up to three-fold simply by washing the harvested cells with distilled water. However this phenomenon was confined to this E. coli strain which excretes the same enzyme into the culture fluid. It was demonstrated that the release of -amylase into the resulting water-wash fraction was not caused by cell lysis but weak osmotic shock. © Rapid Science Ltd. 1998     

Production of poly-β-hydroxybutyrate on molasses by recombinant Escherichia coli

Beet molasses successfully replaced glucose as sole carbon source to produce poly--hydroxybutyrate by a recombinant Escherichia coli strain (HMS174/pTZ18u-PHB). The fermentation with molasses was cheaper than with glucose. The final dry cell weight, PHB content and PHB productivity were 39.5 g/L, 80% (w/w) and 1 g/Lh, respectively, in a 5 L stirred tank fermenter after 31.5 h fed-batch fermentation with constant pH and dissolved O2 content. © Rapid Science Ltd. 1998     

Enhanced production of succinic acid from methanol–organosolv pretreated Strophanthus preussii by recombinant Escherichia coli

Bioprocess and Biosystems Engineering - A biorefinery process for high yield production of succinic acid from biomass sugars was investigated using recombinant Escherichia coli. The major problem...     

Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli

We have previously shown that human interferon α-2b (IFN) produced in Escherichia coli (E. coli) is heterogeneous at the N-terminal, with three major species (Ahsan et al., 2014). These are: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue has been removed by E. coli methionyl aminopeptidase to give the native interferon α-2b and (c) in which the N-terminal Cys residue of the latter contains an acetyl group. In this paper we overcome this heterogeneity, using engineered interferon derivatives with phenylalanine residue directly downstream of the N-terminal methionine (Met-Phe-IFN). This modification not only prevented the removal of the N-terminal methionine by E. coli methionyl aminopeptidase but also the subsequent N-acetylation. Critically, Met-Phe-IFN had enhanced activity in a biological assay. N-terminal stabilization was also achieved by fusing human cytochrome b₅ at the N-terminal of interferon (b₅-IFN-chimera). In this case also, the protein was more active than a reciprocal chimera with cytochrome b₅ at the C-terminal of interferon (Met-IFN-b₅-chimera). This latter protein also had a heterogeneous N-terminal but addition of phenylalanine following Met, (Met-Phe-IFN-b₅-chimera), resolved this problem and gave enhanced biological activity.     

Antitumor effect of combination of murine recombinant interferon β, murine recombinant interferon γ and human recombinant interleukin-2 in MethA-bearing mice

Summary We have previously reported that the combination of murine recombinant interferon (Mu-rIFN) with murine recombinant interferon (Mu-rIFN) provided greater inhibition of tumor growth than did each one alone in MethA-bearing mice. In the present study the effect of addition of human recombinant interleukin-2 (Hu-rIL-2) to the combination of Mu-rIFN with Mu-rIFN on tumor growth in BALB/c mice bearing syngeneic MethA fibrosarcoma was examined. Low doses of Hu-rIL-2 (5 × 10³ U or 5 × 10⁴ U at 3-day intervals) showed no antitumor activity, while a high dose of Hu-rIL-2 (5 × 10⁵ U) showed profound growth inhibition. The administration of IL-2 (ranging between 5 × 10³ U and 5 × 10⁵ U) in addition to the combination of IFN and IFN showed more augmented antitumor effects in a dose-dependent manner. Furthermore, the simultaneous administration of IL-2, IFN and IFN had more effective therapeutic activity, compared with the sequential administration of interferons and IL-2. These findings indicated that IL-2 in combination with IFN and was effective for cancer treatment.     

Identification of a halophilic α-amylase gene from Escherichia coli JM109 and characterization of the recombinant enzyme

A halophilic α-amylase (EAMY) gene from Escherichia coli JM109 was overexpressed in E. coli XL10-Gold and the recombinant protein was purified and characterized. The activity of the EAMY depended on the presence of both Na⁺ and Cl^?, and had maximum activity in 2 M NaCl at 55 °C and pH 7.0. When 2 % (w/v) soluble starch was used as substrate, the specific activity was about 1,090 U mg^?1 protein. This is the first report on identifying a halophilic α-amylase with high specific activity from non-halophilic bacteria.     

Rapid purification of α-lytic protease and its mutants from small cultures of recombinant Escherichia coli

Summary A single-step procedure is described for purifying -lytic protease and its mutants on a small scale (approx. 5mg) from cell-free supernatants of recombinant E. coli. Although free of non-enzyme protein, these preparations often contain fragments that arise from enzymatic self-digestion. Two strong cation exchange matrices are shown to vary greatly in their ability to separate such degradation products from intact enzyme.     

Purification and properties of a novel recombinant human hybrid interferon, σ-4 α2/α1

The human interferon (huIFN) σ-4 α2_5–62/α1_64–166 is a genetically engineered hybrid that consists of residues 5–62 of huIFN α2 and residues 64–166 of huIFN α1. This variant contains four cysteine residues at positions 29, 86, 99 and 139, but does not contain the cysteine at position 1 that is characteristic of naturally occurring huIFN α subtypes. This novel recombinant hybrid was purified fromEscherichia coli to greater than 95% homogeneity. The purification was based on ethanol extraction of a trichloroacetic acid precipitate and Matrex Gel Blue A chromatography followed by either a selective precipitation or DEAE-Sepharose chromatography. The purified protein that was treated with 2-mercaptoethanol exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 17 800 and 17 100, both of which exhibited antiviral activity. Electrophoresis performed without prior reduction with 2-mercaptoethanol indicated only a minor extent of intermolecular disulfide bonding. The purified protein exhibited a high specific antiviral activity of 7·10⁷ units/mg when assayed on human fibroblast cells and, in distinction to the parental huIFN α2, it also demonstrated antiviral activity on murine L929 cells. The level of antiproliferative activity of huIFN δ-4 α2_5–62/α1_64–166 on various cell lines of different histological origin appeared to be more comparable to that of huIFN α1 than huIFN α2. The data suggest that huIFN δ-4 α2_5–62/α1_64–166 hybrid may be a useful tool for understanding huIFN structure-function relations.     

Characterization of a panel of mouse single-chain antibodies against human recombinant interferon β1b

A panel of single-chain Fv-antibodies (ScFv’s) against recombinant human interferon beta 1b (rhIFN-β1b) has been obtained from immune and naïve combinatorial cDNA libraries of the mouse variable immunoglobulin genes. ScFv’s were expressed in Escherichia coli cells. For producers isolated from the immune library a difference in production yield of ScFv’s in periplasm and incubation medium as well as their expression and storage stability have been demonstrated. After sequencing of target DNA the multiple alignment and structural analysis of ScFv’s sequences with different primary structures were carried out and significant difference in both complementarity-determining (CDR) and framework (FR) regions of theirs variable domains has been shown. For the ScFv’s isolated from the immune library, specificity of their binding with native and denatured rhIFN-β1b in ELISA and Western-blotting as well as their high storage stability have been shown. The affinity constants for each representatives of the ScFv’s panel were in the range from 1.96 × 10^?8 to 1.69 × 10^?9 M.     

Salting-out extraction of recombinant κ-carrageenase and phage T7 released from Escherichia coli cells

Traditional technology of cell disruption has become one of the bottlenecks restricting the industrialization of genetic engineering products due to its high cost and low efficiency. In this study, a novel bioprocess of phage lysis coupled with salting-out extraction (SOE) was evaluated. The lysis effect of T7 phage on genetically engineered Escherichia coli expressing κ-carrageenase was investigated at different multiplicity of infection (MOI), meanwhile the phage and enzyme released into the lysate were separated by SOE. It was found that T7 phage could lyse 99.9% of host cells at MOI = 1 and release more than 90.0% of enzyme within 90 min. After phage lysis, 87.1% of T7 phage and 71.2% of κ-carrageenase could be distributed at the middle phase and the bottom phase, respectively, in the SOE system composed of 16% ammonium sulfate and 20% ethyl acetate (w/w). Furthermore, κ-carrageenase in the bottom phase could be salted out by ammonium sulfate with a yield of 40.1%. Phage lysis exhibits some advantages, such as mild operation conditions and low cost. While SOE can efficiently separate phage and intracellular products. Therefore, phage lysis coupled with SOE is expected to become a viable alternative to the classical cell disruption and intracellular product recovery.     

Enhanced γ-aminobutyric acid-forming activity of recombinant glutamate decarboxylase (gadA) from Escherichia coli

γ-aminobutyric acid (GABA) is an important bioactive regulator, and its biosynthesis is primarily through the α-decarboxylation of glutamate by glutamate decarboxylase (GAD). The procedures to obtain GABA by bioconvertion with high activity recombinant Escherichia coli GAD have been seldom understood. In this study, Escherichia coli GAD (gadA) was highly expressed (about 70–75% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-gadA, which was induced by 0.4 mM IPTG in LB medium, and maximal GABA-forming activity of the recombinant GAD was 40 U/mL at a concentration (0.15 mM) of pyridoxal phosphate (PLP) and a concentration (0.6 mM) of Ca²⁺ at optimal pH of 3.8. The optimal concentration (7.5 mM) of Mn²⁺ can also improve the activity of recombinant enzyme, but the co-effect of Ca²⁺ and Mn²⁺ exhibited antagonism effect when added simultaneously. LB and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The relative activity was markedly higher activated by Ca²⁺ (174%), Mn²⁺ (164%) than that by other seven bivalent cations. Finally, the yield of GABA was high of 94 g/L detected by paper chromatography or HPLC in 1 L reaction system with 30 mL crude GAD (12 U/mL). By entrapping Escherichia coli glutamate decarboxylase into sodium alginate and carrageenan gel beads, the activity of immobilized GAD (IGAD) remained 85% during the initial five batches and the activity still remained 50% at the tenth batch, these results indicated that the recombinant Escherichia coli GAD was feasible for the future industrial production of GABA.     

A sandwich-type enzyme-linked immunosorbent assay for the analysis of recombinant human interferon α-2b

The ability of a previously developed sandwich-type enzyme-linked immunosorbent assay (ELISA) for discriminating incorrectly folded recombinant human interferon -2b (IFN-2b) molecular species from multi disulphide-bonded species was investigated. This ELISA was applied to evaluate and improve the effectiveness of the renaturation of IFN-2b, a step that is currently used in the large-scale production of IFN-2b produced in recombinant Escherichia coli strains.     

Construction of a novel recombinant Escherichia coli strain capable of producing 1,3–propanediol and optimization of fermentation parameters by statistical design

Summary The structural gene yqhD from a wild-type Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme and the structural gene dhaB from Citrobacter freundii encoding glycerol dehydratase were amplified by using the PCR method. The temperature control expression vector pHsh harboring the yqhD and dhaB genes was transformed into E. coli JM109 to yield the recombinant strain E. coli JM109 (pHsh-dhaB-yqhD). The response surface method (RSM) was then applied to further optimize the fermentation condition of the recombinant strain. A mathematical model was then developed to show the effect of each medium composition and their interactions on the production of 1,3-propanediol by recombinant strain E. coli JM109. The model estimated that a maximal yield of 1,3-propanediol (43.86 g/l) could be obtained when the concentrations of glycerol, yeast extract and vitamin B₁₂ were set at 61.8 g/l, 6.2 g/l and 49 mg/l, respectively; and the fermentation time was 30 h. These predicted values were also verified by validation experiments. Compared with the values obtained by other runs in the experimental design, the optimized medium resulted in a significant increase in the yield of 1,3-propanediol. The yield and productivity under the optimal parameters and process can reach 43.1 g/l and 1.54 g/l/h. Maximum 1,3-propanediol yield of 41.1 g/l was achieved in a 5-l fermenter using the optimized medium. This makes the engineered strain have potential application in the conversion of glycerol to 1,3-propanediol on an industrial scale.     

Expression of a β-galactosidase gene from Lactobacillus sake in Escherichia coli

Summary A -galactosidase gene from Lactobacillus sake coding for lactose hydrolysis was cloned and expressed in Escherichia coli. Chromosomal DNA from L. sake was partially digested with the restriction enzyme Sau3AI, and the 3–6 Kb fragment was ligated to the cloning vector pSP72 digested with BamHI. One E. coli transformant expressing -galactosidase was isolated on X-gal plates. It contained a plasmid with an insertion of approx. 4 Kb. The restriction map of the recombinant plasmid was constructed. The characteristics of the recombinant -galactosidase were compared with those of the wild type. The optima pH and temperature for both enzymes was 6.5 and 50°C, respectively. Stability of the enzymes at different temperatures and activity on lactose were determined.     

Molecular mass of poly[(R )-3-hydroxybutyric acid] produced in a recombinant Escherichia coli

 Poly[(R)-3-hydroxybutyric acid] (PHB) was produced at 37 °C by a recombinant Escherichia coli harboring the Alcaligenes eutrophus biosynthesis phbCAB genes in Luria-Bertani media containing glucose at 10–30 g/l at different pH values and the time-dependent changes in the molecular mass of PHB were studied. PHB polymers accumulated within cells while glucose was present in the medium. The number-average molecular mass of PHB decreased with time during the course of PHB accumulation, and the values for PHB were markedly dependent on the cultivation conditions of the E. coli, ranging from 0.5 MDa to 20 MDa. Under specific conditions (pH 6.0), E. coli produced PHB with an extremely high molecular mass (20 MDa). It has been suggested that a chain-transfer agent is generated in E. coli cells during the accumulation of PHB. Received: 18 July 1996 / Received revision: 4 November 1996 / Accepted: 4 November 1996     

High-cell-density cultivation of recombinant Escherichia coli,purification and characterization of a self-sufficient biosynthetic octane ω-hydroxylase

We have recently described the biocatalytic characterization of a self-sufficent biosynthetic alkane hydroxylase based on CYP153A13a from Alcanivorax borkumensis SK2 (thereafter A13-Red). Despite remarkable regio- and chemo-selectivity, A13-Red suffers of a difficult-to-reproduce expression and moderate operational stability. In this study, we focused our efforts on the production of A13-Red using high-cell-density cultivation (HCDC) of recombinant Escherichia coli. We achieved 455 mg (5,000 nmol) of functional enzyme per liter of culture. Tight control of cultivation parameters rendered the whole process highly reproducible compared with flask cultivations. We optimized the purification of the biocatalyst that can be performed in either two or three steps depending on the application needed to afford A13-Red up to 95 % homogeneous. We investigated different reaction conditions and found that the total turnover numbers of A13-Red during the in vitro hydroxylation of n-octane could reach up to 3,250 to produce 1-octanol (1.6 mM) over a period of 78 h.     

Enhanced production of human FcγRIIa receptor by high cell density cultivation of Escherichia coli

Human Fc receptors (FcγR) are membrane glycoproteins that are expressed on all immunologically active cells and have a well-defined role in regulating innate and adaptive immune responses by binding to the immunoglobulin G (IgG) antibody. Among the several classes of Fc receptors, FcγRIIa is the most widely expressed, and it serves as an important reagent in antibody engineering. Here, we report on high cell density cultivations (HCDC) of Escherichia coli for preparative scale production of FcγRIIa in a 6.6L bioreactor. Briefly, a pH-stat feeding strategy was employed, and two different cell densities (OD(600) of 46 and 100) were examined for the induction of FcγRIIa gene expression. When cells were induced at a high cell density (OD(600) of 100), the cell density increased to an OD(600) of 234 within 9h after induction, and a 2-fold higher production yield was obtained compared with that of induction at low cell density (OD(600) of 46). After simple purification steps including denaturation and refolding, 87.7 mg of soluble FcγRIIa that was more than 95% pure was obtained from a 20-mL culture with high recovery yield (≈54%). The biological activity of purified FcγRIIa was also confirmed by evaluating its interaction with all subclasses of IgG antibodies using an ELISA bioassay.