title: Expression and purification of untagged full-length HCV NS5B RNA-dependent RNA polymerase.
abstract: The NS5B encoded by the hepatitis C virus genome is a RNA-dependent RNA polymerase essential to viral replication. The entire NS5B protein contains a catalytic domain followed by a regulatory motif and a membrane-anchor domain at its C-terminus. Reported here is the molecular cloning and expression of the full-length NS5B polymerase (NS5B-FL) in bacterial cells as a non-fusion protein. The non-tagged NS5B-FL was purified to homogeneity using sequential chromatographic columns and its identity was confirmed using anti-NS5B peptide antibodies and amino acid sequencing. Purified NS5B-FL demonstrated RNA-dependent RNA polymerase activity and was able to replicate a HCV RNA genome fragment through both copy-back and de novo mechanisms. Its biochemical properties were further characterized in comparison with a truncated form of NS5B polymerase with a deletion of 51 residues from its C-terminus.
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA. qmwang@lilly.com
authors.forename: Q May
authors.initials: QM
authors.lastname: Wang
authors.forename: Robert B
authors.initials: RB
authors.lastname: Johnson
authors.forename: Dayue
authors.initials: D
authors.lastname: Chen
authors.forename: Vincent J-P
authors.initials: VJ
authors.lastname: Lévêque
authors.forename: Jianxin
authors.initials: J
authors.lastname: Ren
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Hockman
authors.forename: Kenji
authors.initials: K
authors.lastname: Abe
authors.forename: Tessho
authors.initials: T
authors.lastname: Hachisu
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kondo
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Isaka
authors.forename: Akihiko
authors.initials: A
authors.lastname: Sato
authors.forename: Tamio
authors.initials: T
authors.lastname: Fujiwara
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase
chemical_list: D017931:DNA Primers; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase