Expression, purification, and characterization of SARS coronavirus RNA polymerase
- 作者：Cheng ; Ao ; Zhang ; Wei ; Xie ; Youhua ; Jiang ; Weihong ; Arnold ; Eddy ; Sarafianos ; Stefan G. ; Ding ; Jianping
- 关键词：SARS ; Coronavirus ; RNA-dependent RNA polymerase ; Polymerization
- 刊名：Virology
- 出版年：2005
- 期刊代码：108_00426822
- 类别：imm
- 出版时间：May 10, 2005
- 卷：335
- 期：2
- 页码：165-176
- 文件大小：1.01 M
摘要
The RNA-dependent RNA polymerase (RdRp) of SARS coronavirus (SARS-CoV) is essential for viral replication and a potential target for anti-SARS drugs. We report here the cloning, expression, and purification of the N-terminal GST-fused SARS-CoV RdRp and its polymerase catalytic domain in Escherichia coli. During purification, the full-length GST-RdRp was found to cleave into three main fragments: an N-terminal p12 fragment, a middle p30 fragment, and a C-terminal p64 fragment comprising the catalytic domain, presumably due to bacterial proteases. Biochemical assays show that the full-length GST-RdRp has RdRp activity and the p64 and p12 fragments form a complex that exhibits comparable RdRp activity, whereas the GST-p64 protein has no activity, suggesting that the p12 domain is required for polymerase activity possibly via involvement in template-primer binding. Nonnucleoside HIV-1 RT inhibitors are shown to have no evident inhibitory effect on SARS-CoV RdRp activity. This work provides a basis for biochemical and structural studies of SARS-CoV RdRp and for development of anti-SARS drugs.