Expression and purification of untagged full-length HCV NS5B RNA-dependent RNA polymerase.
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.
Wang
Q May
QM
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA. qmwang@lilly.com
Johnson
Robert B
RB
Chen
Dayue
D
Lévêque
Vincent J-P
VJ
Ren
Jianxin
J
Hockman
Michelle A
MA
Abe
Kenji
K
Hachisu
Tessho
T
Kondo
Yutaka
Y
Isaka
Yoshitaka
Y
Sato
Akihiko
A
Fujiwara
Tamio
T
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
D017931:DNA Primers; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase