Expression, purification, and partial characterization of HCV RNA polymerase.
The product of the NS5B gene of Hepatitis C Virus (HCV) has been expressed in Escherichia Coli both as a fusion protein with glutathione-S-transferase (GST) of molecular weight 91 KDa and at high level as a single protein of molecular weight 65 KDa. The protein was sequestered within inclusion bodies and a variety of procedures designed to minimize inclusion body formation proved unsuccessful. The method finally adopted involved the purification of inclusion bodies followed by the solubilization, purification, and refolding of the expressed protein. A good recovery and protein purity of the order of 80-90% were achieved. The purified protein was shown to possess RNA polymerase activity in an assay using polyA/oligoU as template. The enzymatic activity is rifampicin resistant, poly A dependent, and requires Mg++. The availability of purified HCV RNA polymerase will allow the study of viral replication and constitute the basis for testing new anti-viral drugs.
Yuan
Z H
ZH
Department of Medicine, St. Mary's Medical School, Imperial College of Science, Technology and Medicine, London, United Kingdom.
Kumar
U
U
Thomas
H C
HC
Wen
Y M
YM
Monjardino
J
J
D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D016174:Hepacivirus / Q000201:enzymology*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism
D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases