Expression, purification, and characterization of SARS coronavirus RNA polymerase.
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.
Cheng
Ao
A
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Zhang
Wei
W
Xie
Youhua
Y
Jiang
Weihong
W
Arnold
Eddy
E
Sarafianos
Stefan G
SG
Ding
Jianping
J
D019380:Anti-HIV Agents / Q000494:pharmacology; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology*; D045169:Severe Acute Respiratory Syndrome / Q000821:virology
D019380:Anti-HIV Agents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D012324:RNA-Dependent RNA Polymerase