Production and characterization of active hepatitis C virus RNA-dependent RNA polymerase

• Published in: Protein expression and purification 2010-06, Vol.71 (2), p.147-152
• Main Authors: Ryu, Kisun, Kim, Kyun-Hwan, Yoo, Seong-Yeon, Lee, Eun-Young, Lim, Keo-Heun, Min, Mi-Kyung, Kim, Hajeong, Choi, Seong Il, Seong, Baik L.
• Format: Article
• Language: English
• Subjects: Animals; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; HCV; Hepacivirus - enzymology; Hepacivirus - genetics; Hepacivirus - metabolism; Hepatitis C virus; High-throughput screening; Kinetics; Lysine-tRNA Ligase - genetics; Lysine-tRNA Ligase - metabolism; LysN fusion; NS5B; Recombinant Proteins - metabolism; RNA Replicase - chemistry; RNA Replicase - genetics; RNA Replicase - metabolism; RNA, Transfer, Amino Acyl - genetics; RNA, Transfer, Amino Acyl - metabolism; RNA-dependent RNA polymerase
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2010.01.004

The non-structural protein 5B (NS5B) is an essential component for the genome replication of hepatitis C virus (HCV). Thus, its activity holds the potential of being a target for therapeutic actions against HCV. The availability of large amount of functionally active NS5B enzyme may facilitate the identification of NS5B inhibitors via high-throughput screening (HTS). Here, we expressed the C-terminal 20-amino acids truncated NS5B in a bacterial system using the N-terminal domain of Escherichia coli lysyl-tRNA synthetase (LysN) as a solubility enhancer. The fusion protein (LysN-NS5B) was purified in a yield of 6.2 mg/L. The activity of LysN-NS5B was confirmed by in vitro RNA-dependent RNA polymerase (RdRp) activity assay, and the biochemical properties of LysN-NS5B were further characterized by kinetic analysis. The optimal RdRp activity was shown at 30 °C with 5 mM of Mg 2+ or 10 mM of Mn 2+, while the K m value for UTP was determined as 5 μM. The RdRp activity of LysN-NS5B was strongly inhibited by phenyldiketoacid, a specific inhibitor of HCV NS5B activity. Our results suggest that the LysN fusion system is a suitable approach for producing an active form of NS5B that can be used for HTS of NS5B inhibitors.