title: [Purification and partial characterization of hepatitis C virus (HCV) non-structural protein 5A (NS5A) expressed in Escherichia coli].
abstract: It has been suggested that non-structural protein 5A (NS5A) of hepatitis C virus (HCV) may have a regulatory role similar to other RNA viruses in RNA replication. In order to investigate the replication function of NS5A, we tried to purify recombinant His(6) tagged NS5A expressed in Escherichia coli by a denature-renaturing method. The native lysis buffer was used to remove most of the soluble non-specific proteins. His-NS5A protein was solublized with the denaturing lysis buffer containing 8 mol/L Urea, and then bound to Ni2+ -NTA resin. The protein bound resin was successively washed with buffer containing reducing concentrations of Urea in the presence of NaCl and DTT to renature the protein. The renatured His-NS5A protein was eluted from the resin and it was capable of interacting with glutathione S-transferase fused form NS5Bt (GST-NS5Bt). The purified His-NS5A exhibited an inhibitory effect on RNA-dependent RNA Polymerase (RdRP) activity of GST-NS5Bt in vitro. Conclusively, in this study, we have established a purification method of bacterial recombinant HCV NS5A, and the results support the notion that NS5A may involve in the regulation of HCV replication through direct interaction with NS5B.
authors.affiliation: Division of Molecular Biology of Infectious Disease, Key Laboratory of Biotherapy of Human Diseases, Ministry of Education, PR China.
authors.forename: Hong
authors.initials: H
authors.lastname: Luo
authors.forename: Weiping
authors.initials: W
authors.lastname: Qin
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Ma
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Shimakami
authors.forename: Seishi
authors.initials: S
authors.lastname: Murakami
authors.forename: Fang
authors.initials: F
authors.lastname: He
authors.forename: Liansan
authors.initials: L
authors.lastname: Zhao
mesh_terms: D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D010957:Plasmids; D011485:Protein Binding; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics; D014779:Virus Replication
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase