The Expression And Purification Of SARS-CoV RNA Dependent RNA Polymerase

The Severe Acute Respiratory Syndrome(SARS) which spread more than twenty countries from Nov.2002 to June 2003 was caused by a novel coronavirus,called SARS-CoV.The complete genome size is 29,727 bp.Phylogenetic study indicates that SARS-CoV may stand up as the forth group of coronavirus.The main viral proteins are S, E,M,N,3CL,and RDRP.Coronavirus replication in cells is initiated by translation of the two overlapping open reading frames(ORF1a and ORF1b) of the replicase gene to yield two polyproteins, ppla and pplab.The replicase polyproteins is predicted to be mediated by two virus-encoded proteinases,a 3C-like proteinase(3CLpro) and a papain-like proteinase (PLP).The SARS-CoV replicase gene encodes 16 nsps with multiple enzymatic functions,included RNA-dependent RNA polymerase activity(RDRP,nsp12). Coronavirus nsp12-RdRp is believed to play a central role in both replication and transcription.RDRP is the uniquely defined non-structural protein responsible for RNA replication,mutation rate or fidelity,regulation of transcription in coronaviruses and many other ssRNA viruses.The aim of this study is to observe RDRP expression and subcellular localization in 293 cells.The other aim is to express and purify the wild and mutant RDRP fusion protein in E.coli.Firstly,we have cloned the full-length of the CDS part of RDRP of SARS-CoV.Secondly,RDRP was cloned into pCMV-Myc vector and pGEX-4T-1 vector. And,to determine whether GDD motif is the important domain for the biological activity of RDRP,the following approaches were employed;1) PCR site-directed mutagenesis was used to construct eukaryotic expression and prokaryotic expression vector of GDD→ADD mutants,respectively,and named pCMV-Myc-M-Pol and pGEX-4T-1-M-Pol;2) The plasmid pCMV-Myc-Pol was transiently transfected in to 293cell.The expression and localization of RDRP protein was observed by immunostaining approach.The wild and mutant GST-RDRP fusion protein was expressed in E.coli and purified by GST affinity chromatography.In this study,we constructed the eukaryotic and prokaryotic RDRP protein expression vector pCMV-Myc-Pol,pCMV-Myc-M-Pol and pGEX4T-1-Pol,pGEX4T-1-M-Pol defined the subcellular location of RDRP protein,and purified both wild and mutant GST-RDRP fusion protein from E.coli.We found that the molecule weight of RDRP protein expressed in eukaryote versus prokaryote is different,indicating that a posttranslational modification may occur during RDRP protein synthesis in the cell.In addition,the purified RDRP protein from bacteria may be useful for the production of specific antibody against RDRP protein in the future.CONCLUSION:RDRP protein was highly expressed in transfected 293 cells,and distributed in cytoplasm.The wild and mutant GST-RDRP fusion protein was expressed in E.coli cells.And both the wild and mutant recombinant GST-RDRP fusion protein had been successfully purified from E.coli cells,and could be used for further antibody production and functional characterization.