Optimization of conditions for high‐level expression and purification of human recombinant consensus interferon (rh‐cIFN) and its characterization

Abstract

Recombinant human consensus interferon (rh-cIFN) is an artificially engineered interferon (IFN) developed by recombining and reordering the protein sequences that exist in standard IFN. This recombination resulted into a drug that has the potential to work better than natural, standard IFN. In this study, we described optimized conditions for high-level expression and recovery of biologically active consensus IFN from inclusion bodies (IBs). A synthetic gene coding 166 amino acids of consensus IFN was cloned under the T7 promoter. Escherichia coli strain BL21DE3Plys was used to transform expression construct. For high-level expression, shake-flask fermentation conditions were standardized. For isolation of IBs, the sonication method was optimized. A variety of chaotropic agents including guanidine hydrochloride, urea, SDS, and detergents were studied for solubilization of IBs. For renaturation of solubilized denatured protein by the dilution process, parameters of dilution factor, temperature, and l-arginine were optimized. A one-step chromatography method was developed for high-yield purification of consensus IFN. rh-cIFN was characterized by SDS-PAGE, Western blot, and high-performance liquid chromatography. Purified protein has a molecular weight of 19.5 kDa and specific activity was 2.0 × 10(8) as determined by the cytopathic inhibition assay. This study concludes that by using optimized conditions, we obtained a yield of 100 mg/L of biologically active rh-cIFN, which is highest ever reported according to available data.

Strategies for the Production of Soluble Interferon-Alpha Consensus and Potential Application in Arboviruses and SARS-CoV-2

“…Even though there is no massive data about cIFN purification from E. coli , recombinant IFN-α is also produced as inclusion bodies, refolded, and recovered. Many authors related their experience in purifying IFN-α from inclusion bodies mainly using Q-Sepharose [ 71 , 72 , 73 ] and DEAE Sepharose [ 28 , 74 ]. While we were not able to recover pure cIFN from HD-IFN molecule, the untagged cIFN obtained from HF-IFN showed 100% purity by HPSEC after TEV hydrolysis, with a yield of 8 mg/L.…”

Production of a bioactive recombinant chicken matrix metalloproteinase‐11 peptide in Escherichia coli

“…In the present study, prokaryotic expression of the truncated cMMP‐11 peptide using pET32b(+) vector and different strains of E. coli revealed expression of the peptide as inclusion bodies at temperatures ranging from 28 to 37 °C. Recently, a study involving pET vector and E. coli BL21(DE3)pLys also reported very high level expression of the recombinant protein as inclusion bodies . Among the strains, mean percentage intensity of the recombinant peptide was found to be the highest in E .…”

Enhancing recombinant interleukin‐6 production yield by fermentation optimization, two‐step denaturing, and one‐step purification

“…At this point, the culture was induced with a final concentration of 1 mM IPTG and incubated for a further 16 H. In method M2, fermentation of the rhIL‐6 in the shake flask was performed as described by Ahmed et al. . Clones with the high‐level expression of rhIL‐6 as a preculture were grown at 37 °C in 100 mL supplemented with 34 mg/mL chloramphenicol and 50 mg/mL kanamycin and used to inoculate 1 L of LB broth (in a 2‐L baffled flask).…”

“…Refolding of the protein was carried out at 20 °C for 18 H by the simple dilution method. Denatured protein was added dropwise into the refolded buffer (100 mM Tris–HCl, pH 8.5, 0.5 M l ‐arginine, cysteine/cysteine 90 mg/50 mg, 8 mM EDTA, pH 8.0) with continuous stirring . To remove the any visible precipitation in the refolded protein, the solution was passed through 0.45 μm filter.…”