title: Production and characterization of active hepatitis C virus RNA-dependent RNA polymerase.
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 6.2mg/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 degrees C with 5mM of Mg(2+) or 10mM of Mn(2+), while the K(m) value for UTP was determined as 5microM. 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.
authors.affiliation: Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea.
authors.forename: Kisun
authors.initials: K
authors.lastname: Ryu
authors.forename: Kyun-Hwan
authors.initials: KH
authors.lastname: Kim
authors.forename: Seong-Yeon
authors.initials: SY
authors.lastname: Yoo
authors.forename: Eun-Young
authors.initials: EY
authors.lastname: Lee
authors.forename: Keo-Heun
authors.initials: KH
authors.lastname: Lim
authors.forename: Mi-Kyung
authors.initials: MK
authors.lastname: Min
authors.forename: Hajeong
authors.initials: H
authors.lastname: Kim
authors.forename: Seong Il
authors.initials: SI
authors.lastname: Choi
authors.forename: Baik L
authors.initials: BL
authors.lastname: Seong
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008250:Lysine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D012346:RNA, Transfer, Amino Acyl; D011994:Recombinant Proteins; D012324:RNA-Dependent RNA Polymerase; D008250:Lysine-tRNA Ligase