Over-expression and characterization of NS3 and NS5A of Hepatitis C virus genotype 3a

Microbial Cell Factories

Research

Mohammad S Yousef¹  Muhammad Ikram Anwar²  Moazur Rahman²  Mazhar Iqbal² 

[1] Department of Physics, College of Arts and Sciences, Southern Illinois University, 62026-1654, Edwardsville, Edwardsville, IL, USA;Biophysics Department, Faculty of Science, Cairo University, 12613, Giza, Egypt;Health Biotechnology Division, Drug Discovery and Structural Biology group, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan;

关键词: HCV;    NS3;    NS5A;    Genotype 3a;    Protease;    Helicase;    Viral replication;    Protein expression;    CD spectroscopy;    FT-IR;   

DOI  :  10.1186/1475-2859-12-111

 received in 2013-07-24, accepted in 2013-11-12,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundHepatitis C virus (HCV) is a common and leading cause for liver cirrhosis and hepatocellular carcinoma. Current therapies to treat HCV infection are shown to be partially effective and poorly tolerated. Therefore, ample efforts are underway to rationally design therapies targeting the HCV non-structural proteins. Most of the work carried out in this direction has been focusing mainly on HCV genotype 1. Two direct-acting antiviral agents (DAAs) Telaprevir and Boceprevir are being used against genotype 1a infection in combination therapy with interferon and ribavirin. Unfortunately these DAAs are not effective against genotype 3a. Considering the wide spread infection by HCV genotype 3a in developing countries especially South Asia, we have focused on the recombinant production of antiviral drug targets NS3 and NS5A from HCV genotype 3a. These protein targets are to be used for screening of inhibitors.ResultsHigh-level expression of NS3 and NS5A was achieved at 25°C, using ~1 and 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), respectively. Yields of the purified NS3 and NS5A were 4 and 1 mg per liter culture volume, respectively. Although similar amounts of purified NS3 were obtained at 25 and 14°C, specificity constant (Kcat/Km) was somewhat higher at expression temperature of 25°C. Circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopy revealed that both NS3 and NS5A contain a mixture of alpha-helix and beta-sheet secondary structures. For NS3 protein, percentages of secondary structures were similar to the values predicted from homology modeling.ConclusionsNS3 and NS5A were over-expressed and using Nickel-affinity method both proteins were purified to ~ 95% purity. Yield of the purified NS3 obtained is four fold higher than previous reports. CD spectroscopy revealed that difference in activity of NS3 expressed at various temperatures is not related to changes in global structural features of the protein. Moreover, CD and FT-IR analysis showed that NS3 and NS5A contain both alpha-helical and beta-sheet structures and for NS5A, the proportion is almost equal. The production of NS3 and NS5A in milligram quantities will allow their characterization by biophysical and biochemical means that will help in designing new strategies to fight against HCV infection.

【 授权许可】

Unknown   
© Anwar et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.