Author: Fan, Zheng; Peng, Kunpeng; Tan, Xinyu; Yin, Bin; Dong, Xiuhua; Qiu, Feichan; Shen, Yan; Wang, Heng; Yuan, Jiangang; Qiang, Boqin; Peng, Xiaozhong
Title: Molecular cloning, expression, and purification of SARS-CoV nsp13 Cord-id: epqbljzn Document date: 2005_4_11
ID: epqbljzn
Snippet: 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 0.5 mM IPTG and purified by a single Ni(2+) affinity chromatography. The p
Document: 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 0.5 mM 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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