Molecular cloning, expression, and purification of SARS-CoV nsp13 |
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Authors: | Fan Zheng Peng Kunpeng Tan Xinyu Yin Bin Dong Xiuhua Qiu Feichan Shen Yan Wang Heng Yuan Jiangang Qiang Boqin Peng Xiaozhong |
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Affiliation: | National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China. |
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Abstract: | The SARS-nsp13 protein was identified as an mRNA cap1 methyltransferase. In this study, the nsp13 gene was cloned from the SARS-CoV PUMC02 strain viral RNA by RT-PCR, and inserted into the expression plasmid pET30a(+). The recombinant plasmid pET30a(+)-nsp13 was confirmed by restriction enzymes and sequencing analysis, and transformed into Escherichia coli BL21(DE3). The His-tag-fused protein was expressed by induction of 0.5mM IPTG and purified by a single Ni(2+) affinity chromatography. The protein was validated by western blot and MS analysis. A large quantity of the nsp13 protein obtained with this method may be useful for further study of its structure and function. |
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