Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Purification and Characterization of Recombinant Hepatitis C Virus Replicase

  • Park, Chan-Hee (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Kee, Young-Hoon (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Lee, Jong-Ho (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Oh, Jang-Hyun (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Park, Jung-Chan (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Myung, Hee-Joon (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)
  • Published : 1999.12.01
Download PDF
⟨ Previous Next ⟩

Abstract

The gene encoding the RNA-dependent RNA polymerase of the hepatitis C virus was cloned and expressed with a C-terminal hexahistidine tag. The protein was purified from Escherichia coli to near homogeneity and characterized in vitro. When the 21 amino acids from the C-terminus of the protein were deleted, an inclusion body was not formed and a better purification yield was achieved. However, the activity of the purified enzyme decreased compared to that of the full length protein. The purified enzyme did exhibit ribonucleotide-incorporation activity on an in vitro transcribed RNA containing the 3' end of the HCV genome. It also possessed ribonucleotide incorporation activity, to a lesser extent, on in vitro transcribed foreign RNA templates when RNA or DNA primers were present. The activity was higher with DNA primers than with RNA primers. Accordingly, this assay system will facilitate the screening of inhibitors for hepatitis C virus replication.

Keywords

References

  1. Virus Res. v.53 Expression of recombinant hepatitis C virus non-structural protein 5B in Escherichia coli Al, R.;Y. Xie;Y. Wang;C. Hagedorn
  2. Antiviral Chem. Chemotherapy v.8 Molecular targets in inhibition of hepatitis C virus replication Bartenschlager, R.
  3. EMBO J. v.15 Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus Behrens, S.-E.;L. Tomei;R. De Francesco
  4. Science v.244 Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome Choo, Q.-L.;G. Kuo;A. Weiner;L. R. Overby;D. W. Bradley;M. Houghton
  5. Proc. Nat. Acad. Sci. USA v.88 Genetic organization and diversity of the hepatitis C virus Choo, Q.-L;K. H. Richman;J. H. Han;K. Berger;C. Lee;C. Dong;C. Gallegos;D. Coit;A. Medina-Selvy;P. J. Barr;A. J. Weiner;D. W. Bradley;G. Kuo;M. Houghton
  6. J. Virol. v.69 An amino terminal domain of the hepatitis C virus NS3 protease is essntial for interactio with NS4A Failla, C.;Tomei;R. De Francesco
  7. J. Virol. v.73 Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli Ferrari, E.;J. Wright-Minogue;J. Fang;B. Baroudy;J. Lau;Z. Hong
  8. Arch. Virol. Suppl. v.2 Classification and nomenclature of viruses Frankie, R. I.;D. L. Faquet;D. L. Knudson;F. Brown
  9. J. Microbiol. Biotechnol. v.8 Expression and characterization of recombinant E2 protein of hepatitis C virus by insect cell/baculovirus expression system Han, B. K.;B. Lee;M. K. Min;K. Jung
  10. Hepatology v.29 Expression of hepatitis C virus NS5B protein: Characterization of its RNA polymerase activity and RNA binding Ishii, K.;Y. Tanaka;C. Yap;H. Aizaki;Y. Matsuura;T. Miyamura
  11. Science v.285 Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line Lohman, V.;F. Korner;J.-O. Koch;U. Herian;L. Theilmann;r. Bartenschlager
  12. Virology v.249 Biochemical and kinetic analysis of NS5B RNA-dependent RNA polymerase of the hepatitis C virus Lohman, V.;A. Roos;F. Korner;J. Koch;R. Bartenschlager
  13. J. Biol. Chem. v.273 RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region Yamachita, T.;S. Kanenko;Y. Shirota;W. Qin;T. Nomura;K. Kobayashi;S. Murakami
  14. Biochem. Biophys. Res. Commun. v.232 Expression, purification, and partial characterization of HCV RNA polymerase Yuan, Z.;U. Kumar;H. Thomas;Y.-M. Wen;J. Monjardino