Molecular cloning, expression, and purification of SARS-CoV nsp13

Zheng Fan; Kunpeng Peng; Xinyu Tan; Bin Yin; Xiuhua Dong; Feichan Qiu; Yan Shen; Heng Wang; Jiangang Yuan; Boqin Qiang; Xiaozhong Peng

doi:10.1016/j.pep.2004.08.003

Molecular cloning, expression, and purification of SARS-CoV nsp13

Protein Expr Purif. 2005 Jun;41(2):235-40. doi: 10.1016/j.pep.2004.08.003.

Authors

Zheng Fan¹, Kunpeng Peng, Xinyu Tan, Bin Yin, Xiuhua Dong, Feichan Qiu, Yan Shen, Heng Wang, Jiangang Yuan, Boqin Qiang, Xiaozhong Peng

Affiliation

¹ 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.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Chromatography, Affinity
Cloning, Molecular*
Escherichia coli / genetics
Gene Expression Regulation, Viral
Genetic Vectors / genetics
Methyltransferases / biosynthesis*
Methyltransferases / genetics*
Methyltransferases / isolation & purification
Molecular Sequence Data
RNA Helicases
RNA, Viral / genetics
Recombinant Fusion Proteins / biosynthesis
Recombinant Fusion Proteins / genetics
Reverse Transcriptase Polymerase Chain Reaction / methods
Severe acute respiratory syndrome-related coronavirus / enzymology*
Severe acute respiratory syndrome-related coronavirus / genetics*
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods
Viral Nonstructural Proteins
Viral Proteins / biosynthesis
Viral Proteins / genetics
Viral Proteins / isolation & purification

Substances

RNA, Viral
Recombinant Fusion Proteins
Viral Nonstructural Proteins
Viral Proteins
Methyltransferases
Nsp13 protein, SARS-CoV
RNA Helicases