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

Nadeem Ahmed, Hamid Bashir, Ahmad Usman Zafar, Mohsin Ahmad Khan, Saad Tahir, Faidad Khan, Muhammad Islam Khan, Muhammad Akram, Tayyab Husnain

Biotechnology and applied biochemistry 2015 Sep-Oct

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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. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

Citation

Nadeem Ahmed, Hamid Bashir, Ahmad Usman Zafar, Mohsin Ahmad Khan, Saad Tahir, Faidad Khan, Muhammad Islam Khan, Muhammad Akram, Tayyab Husnain. Optimization of conditions for high-level expression and purification of human recombinant consensus interferon (rh-cIFN) and its characterization. Biotechnology and applied biochemistry. 2015 Sep-Oct;62(5):699-708