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

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

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

Bibliographical note

Funding Information:
We thank Moon-Suhn Ryu, Food Science and Human Nutrition Department, University of Florida, for helpful discussion and preparation of the manuscript. The work has been supported in part by the National Strategic Research Grant from the Ministry of Knowledge Economy of the Korean Government (Grant No. 10031969 ) and the National Research Foundation of Korea Grant funded by the Korean Government (MEST) ( 2009-0092970 ). This work was also supported in part by the Yonsei University Research Fund of 2009 (to K. Ryu and S.I. Choi).

All Science Journal Classification (ASJC) codes

  • Biotechnology

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