Refolding of recombinant human interferon ��-2a from Escherichia coli by urea gradient size exclusion chromatography

Protein refolding is still a puzzle in the production of recombinant proteins expressed as inclusion bodies (IBs) in Escherichia coli. Gradient size exclusion chromatography (SEC) is a recently developed method for refolding of recombinant proteins in IBs. In this study, we used a decreasing urea gradient SEC for the refolding of recombinant human interferon ??-2a (rhIFN??-2a) which was overexpressed as IBs in E. coli. In chromatographic process, the denatured rhIFN??-2a would pass along the 8.0?C3.0 M urea gradient and refold gradually. Several operating conditions, such as final concentration of urea along the column, gradient length, the ratio of reduced to oxidized glutathione and flow rate were investigated, respectively. Under the optimum conditions, 1.2 × 10⁸ IU/mg of specific activity and 82% mass recovery were obtained from the loaded 10 ml of 1.75 mg/ml denatured protein, and rhIFN??-2a was also purified during this process with the purity of higher than 92%. Compared with dilution method, urea gradient SEC was more efficient for the rhIFN??-2a refolding in terms of specific activity and mass recovery.     

Over-expression of recombinant human phospholipid scramblase 1 in E. coli and its purification from inclusion bodies

Human phospholipid scramblase 1 (hPLSCR1) scrambles plasma membrane phospholipids during cell activation, blood coagulation and apoptosis. It was over-expressed in E. coli with a histidine tag and purified from the inclusion bodies (~30 mg/l culture broth) under denaturing conditions using 8 M urea. The denatured hPLSCR1 refolded into its native configuration when urea was removed as shown by a 10-fold increase in its intrinsic fluorescence. Active hPLSCR1 showed scrambling activity in vitro after reconstituting in proteoliposomes. hPLSCR1 showed higher rates of scrambling activity for phosphatidylethanolamine than phosphatidylcholine. Binding studies with the calcium analogue “Stains-all” dye showed a characteristic peak, termed as the J band, at 650 nm. This is the first report on high level expression of hPLSCR1 with histidine tag in E. coli.     

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.     

Optimization of soluble human interferon-γ production in Escherichia coli using SUMO fusion partner

Interferon-γ (IFN-γ) is a broad-spectrum antiviral glycoprotein that produced by lymphatic T cells and natural killer cells those who had stimulated by antigen. Human IFN-γ (hIFN-γ) often used in clinical research and practice because of its bioactivity, for example, antivirus, antitumor, controlling cell apoptosis, and the strict selectivity. However, due to the difficulties of Escherichia coli expression system meet in protein folding, the hIFN-γ often existed as inclusion body. The production of soluble hIFN-γ can be developed to shorten the production cycle and decrease the cost. In this study, small ubiquitin-related modifier fusion technology was used to express and purify recombinant hIFN-γ. Expression induced by adding 50 mM arginine and 1 % (w/v) glycerol into the culture at 24 °C existed as a soluble form of 70 % in total protein. Finally, about 62 mg recombinant hIFN-γ was obtained from 1 L fermentation culture with no less than 96 % purity. Determined by cytopathic effect inhibition assay, the specific activity of the recombinant hIFN-γ achieved at 7.78 × 10⁵ IU/mL.     

Optimization of conditions for expression of recombinant interferon-γ in E.coli

Interferon gamma (IFN-γ) is an important immunoregulatory cytokine that has a central role against viral and bacterial infections. In this study, the cDNA encoding 141 amino acids of mature IFN-γ from mice splenocytes was cloned in a prokaryotic expression vector pQE 30. Optimization of expression conditions resulted in high IFN-γ protein. Western blot showed that recombinant IFN-γ was specifically recognized by its counterpart anti-mouse IFN-γ antibodies. In vitro dose-dependent studies, with A549 and HeLa cell lines, showed that cloned IFN-γ was safe and had no effect on cell proliferation. The protein prediction and analysis using SOPMA program, revealed that IFN-γ had 80 α-helices, 8 β-turns jointed by 9 extended strands and 44 random coils. A total of four major clusters were observed with murine IFN-γ sharing 39 % homology with human IFN-γ. Pair-wise alignment studies with human revealed 26 % identity and 43.3 % similarity. The recovery of bioactive proteins from inclusion bodies (IBs) is a complex process and various protocols have been developed. We report here a simple, robust and inexpensive purification approach for obtaining recombinant IFN-γ protein expressed as IBs in E.coli.     

Production of interferon-α in high cell density cultures of recombinant Escherichia coli and its single step purification from refolded inclusion body proteins

Escherichia coli TG1 transformed with a temperature-regulated interferon-α expression vector was grown to high cell density in defined medium containing glucose as the sole carbon and energy source, utilizing a simple fed-batch process. Feeding was carried out to achieve an exponential increase in biomass at growth rates which minimized acetate production. Thermal induction of such high cell density cultures resulted in the production of ∼4 g interferon-α/l culture broth. Interferon-α was produced exclusively in the form of insoluble inclusion bodies and was solubilized under denaturing conditions, refolded in the presence of arginine and purified to near homogeneity, utilizing single-step ion-exchange chromatography on Q-Sepharose. The yield of purified interferon-α was ∼300 mg/l with respect to the original high cell density culture broth (overall yield of ∼7.5% active interferon-α). The purified recombinant interferon-α was found by different criteria to be predominantly monomeric and possessed a specific bioactivity of ∼2.5 × 10⁸ IU/mg based on viral cytopathic assay. Received: 8 October 1999 / Received revision: 8 December 1999 / Accepted: 12 December 1999     

Separation and purification of Escherichia coli-expressed human thymosin-α1 using affinity chromatography and high-performance liquid chromatography

In this study, a human thymosin-α1 (hTα1) fusion protein was overexpressed in Escherichia coli (E. coli). The hexahistidine-tagged hTα1 fusion protein was obtained in soluble form in cells of the engineered E. coli strain BL21 (DE3)/pET-28a-hTα1 that had been induced with isopropyl -D-1-thiogalactopyranoside (IPTG). The recombinant protein accounted for approximately 50-60% of the total protein. We then developed and validated a separation method for hTα1 from E. coli cells based on thermal denaturation, nickel-resin affinity chromatography and high-performance liquid chromatography. The purification method showed good reproducibility and was easy to operate. Purified recombinant hTα1 of high homogeneity was characterized and found to be of high purity (over 99%), as determined by high-voltage electrophoresis and high-performance liquid chromatography analysis. Isoelectric focusing analysis indicated a pI of approximately 4.0, and full wavelength screening showed an optimal absorbance wavelength at around 214nm.     

Effect of lipid supplements on the production and glycosylation of recombinant interferon-γ expressed in CHO cells

The effects of lipids on the glycosylation of recombinant human interferon- expressed in a Chinese Hamster Ovary cell line were investigated in batch culture. Lipids form an essential part of the N-glycosylation pathway, and have been shown to improve cell viability. In control (serum-free) medium the proportion of fully-glycosylated interferon- deteriorated reproducibly with time in batch culture, but the lipoprotein supplement ExCyte was shown to minimise this trend. Partially substituting the bovine serum albumin content of the medium with a fatty-acid free preparation also improved interferon- glycosylation, possibly indicating that oxidised lipids carried on Cohn fraction V albumin may damage the glycosylation process.Abbreviations BSA bovine serum albumin - CHO chinese hamster ovary - DHFR dihydrofolate reductase - FCS foetal calf serum - IFN- human interferon-gamma - q _IFN specific interferon production rate - specific growth rate - 2N doubly-gycosylated - 1N singly-glycosylated - ON non-glycosylated     

Purification and characterization of prokaryotically expressed human interferon-λ2

A system for the production of soluble interferon (IFN)-λ2 was developed by fusing the IFN-λ2, NusA protein, polyhistidine and S peptide genes and then expressing the fused product (Nus-His-S-tagged IFN-λ2) in Escherichia coli. The expressed fusion protein was purified by Ni-NTA affinity chromatography. The fusion tag was removed from recombinant IFN-λ2 by cleavage with enterokinase. N-Terminal sequencing confirmed the identity of the purified protein. When compared with commercial IFN-α2b, IFN-λ2 had similar antiviral activity. The production and characterization of biologically active IFN-λ2 will be beneficial for its potential role in clinical applications.     

Production and purification of recombinant human α2C2 adrenergic receptor using Saccharomyces cerevisiae

The objective is to generate milligram quantities of recombinant human ₂C2 adrenergic receptor for X-ray crystallographic studies. It has been cloned in Saccharomyces cerevisiae, and the production level is at best about 13 pmol/mg of membrane protein, as estimated by radio-ligand binding assay. The receptor is solubilized with sucrose monolaurate followed by immunoaffinity purification and reconstitution into phospholipid vesicles. The efficiency of solubilization and immuno-purification are 60% and 91%, respectively.     

A semi-continuous process for the production of human interferon-αc from E. coli using tangential-flow microfiltration and immuno-affinity chromatography

Summary A semi-continuous process for the production of human interferon-c (IFN) from E. coli is described. Harvesting of bacteria, removal of cell debris and concentration were performed by tangentialflow microfiltration (cross-flow microfiltration). Purification was done in a single step by a monoclonal-Ab column. In the process, purified material, 3–9×10⁸ units/mg protein was obtained with a 65% overall yield.     

Monitoring proteolysis of recombinant human interferon-γ during batch culture of Chinese hamster ovary cells

Proteolytic cleavage of recombinant human interferon- (IFN-) expressed in Chinese hamster ovary (CHO) cells during batch fermentation has been monitored by mass spectrometric peptide mapping. IFN- was purified from cell-free culture supernatant by immunoaffinity chromatography and cleaved by endoprotease Asp-N. Peptide fragments were resolved by reverse-phase HPLC and identified by a combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and automated N-terminal peptide sequencing. Using this approach, a peptide was identified as the C-terminal fragment of the IFN- polypeptide. Analysis of this peptide by MS indicated that the recombinant IFN- polypeptide secreted by CHO cells was truncated by at least ten amino acids, initially at Gln¹³³-Met¹³⁴. No full length (143 amino acids) polypeptide molecules were observed at any stages of the fermentation. Additional proteolytic cleavages at basic amino acids N-terminal of Gln¹³³ occurred during the later stages of the culture resulting in a heterogeneous IFN- polypeptide population with 'ragged' C-termini.     

Expression and purification of moricin CM4 and human β-defensins 4 in Escherichia coli using a new technology

Different strategies have been developed to produce small antimicrobial peptides using recombinant techniques. Here we report a new technology of biosynthesis of moricin CM4 and human β-defensins 4 (HβD4) in the Escherichia coli. The CM4 and HβD4 gene were cloned into a vector containing the tags elastin-like peptide (ELP) and intein to construct the expression vector pET-EI-CM4 and pET-EI-HβD4. All the peptides, expressed as soluble fusions, were isolated from the protein debris by the method called inverse transition cycling (ITC) rather than traditional immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by self-cleavage. Fully reduced peptides that were purified exhibited expected antimicrobial activity. The approach described here is a low-cost, convenient and potential way for generating small antimicrobial peptide.     

Expression of recombinant human interferon-γ with antiviral activity in the bi-cistronic baculovirus-insect/larval system

A bi-cistronic baculovirus-insect/larval system containing a polyhedron promoter, an internal ribosome entry site (IRES), and an egfp gene was developed as a cost-effective platform for the production of recombinant human interferon gamma (rhIFN-γ). There was no significant difference between the amounts of rhIFN-γ produced in the baculovirus-infected Spodoptera frugiferda 21 cells grown in serum-free medium and the serum-supplemented medium, while the Trichoplusia ni (T. ni) and Spodoptera exigua (S. exigua) larvae afforded rhIFN-γ amounting to 1.08±0.04 and 9.74±0.35 μg/mg protein respectively. The presence of non-glycosylated and glycosylated rhIFN-γ was confirmed by immunoblot and lectin blot. The immunological activity of purified rhIFN-γ, with 96% purity by Nickel (II)-nitrilotriacetic acid (Ni-NTA) affinity chromatography, was similar to that commercially available. Moreover, the rhIFN-γ protein from T. ni had more potent antiviral activity. These findings suggest that this IRES-based expression system is a simple and inexpensive alternative for large-scale protein production in anti-viral research.     

Na-butyrate increases the production and α2,6-sialylation of recombinant interferon-γ expressed by α2,6- sialyltransferase engineered CHO cells

A non-human like glycosylation pattern in human recombinant glycoproteins expressed by animal cells may compromise their use as therapeutic drugs. In order to correct the CHO glycosylation machinery, a CHO cell line producing recombinant human interferon- (IFN) was transformed to replace the endogenous pseudogene with a functional copy of the enzyme 2,6-sialyltransferase (2,6-ST). Both the parental and the modified CHO cell line were propagated in serum-free batch culture with or without 1 mM sodium butyrate. Although Na-butyrate inhibited cell growth, IFN concentration was increased twofold. The IFN sialylation status was determined using linkage specific sialidases and HPLC. Under non- induced conditions, IFN expressed by 2,6-engineered cells contained 68% of the total sialic acids in the 2,6- conformation and the overall molar ratio of sialic acids to IFN was 2.3. Sodium butyrate addition increased twofold the molar ratio of total sialic acids to IFN and 82% of total sialic acids on IFN were in the 2,6-conformation. In contrast, no effect of the sodium butyrate was noticed on the sialylation of the IFN secreted by the 2,6-ST deficient parental cell line. This study deals for the first time with the effect of Na-butyrate on CHO cells engineered to produce human like sialylation.     

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.     

Process development for production of recombinant human interferon-γ expressed in<Emphasis Type="Italic"> Escherichia coli</Emphasis>

A simple fed-batch process was carried out using constant and variable specific growth rates for high-cell-density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-(hIFN-). The feeding rate was adjusted to achieve an appropriate specific growth rate. The dissolved oxygen level was maintained at 20–30% of air saturation by control of airflow and stirrer speed and, where necessary, by enrichment of inlet air with pure oxygen. Glucose was the sole source of carbon and energy and was provided by following a simple exponential feeding rate. The final cell density in the fed-batch fermentation with constant and variable specific growth rate feeding strategies was ~100 g dry cell wt l^–1 after 36 and 20 h, respectively. The final specific yield and overall productivity of recombinant hIFN- in the variable specific growth rate strategy were 0.35 g rHu-IFN- g^–1 dry cell wt and 0.9 g rHu-IFN- l^–1 h^–1, respectively. A new chromatographic purification procedure involving anion exchange and cation exchange chromatographies was developed for purification of rHu-IFN- from inclusion bodies. The established purification process is reproducible and the total recovery of rHu-IFN- was ~30% (100 mg rHu-IFN- g^–1 dry cell wt). The purity of the rHu-IFN- was determined using HPLC. Sterility, pyrogenicity, and DNA content tests were conducted to assure the absence of toxic materials and other components of E. coli in the final product. The final purified rHu-IFN- has a specific antiviral activity of ~2×10⁷ IU/mg protein, as determined by viral cytopathic effect assay. These results certify the product for clinical purposes.     

CHO cell growth and recombinant interferon-γ production: Effects of BSA, Pluronic and lipids

The role of bovine serum albumin in mammalian cell cultures and the possibility of its substitution by other components in a serum-free medium has been investigated. In this study, BSA was shown to be important for growth and product formation in CHO cells expressing recombinant human interferon-. There were indications that its stimulating growth effect was dependent on the source of BSA used and probably was related to the purification procedure used for the production of the desired albumin fraction. Cell growth did not occur in the absence of BSA but at low concentration (1 mg ml^–1) it was stimulated by the addition of a combination of a commercial lipid mixture plus Pluronic F68. However, under the latter conditions IFN- production was adversely effected. The importance of individual lipid components was investigated using a statistical approach based on a Plackett-Burman design. Linoleic acid was identified as a positive variable for cell growth while cholesterol was identified as a negative variable for both cell growth and IFN- production. When a combination of linoleic acid plus Pluronic F68 was included in the formulation of low BSA medium, cell growth was similar to that at high BSA concentration (5 mg ml^–1) but the IFN- concentration was significantly reduced (ca. 45%).Abbreviations IFN- interferon- - CHO Chinese Hamster Ovary cells - BSA bovine serum albumin - FAF-BSA fatty acid-free bovine serum albumin