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

Kisun Ryu, Kyun Hwan Kim, Seong Yeon Yoo, Eun Young Lee, Keo Heun Lim, Mi Kyung Min, Hajeong Kim, Seong Il Choi, Baik L. Seong

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Abstract

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 Mg2+ or 10 mM of Mn2+, while the Km 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.

Original languageEnglish
Pages (from-to)147-152
Number of pages6
JournalProtein Expression and Purification
Volume71
Issue number2
DOIs
Publication statusPublished - 2010 Jun 1

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All Science Journal Classification (ASJC) codes

  • Biotechnology

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