Molecular cloning, expression, and purification of SARS-CoV nsp13.
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.
Fan
Zheng
Z
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.
Peng
Kunpeng
K
Tan
Xinyu
X
Yin
Bin
B
Dong
Xiuhua
X
Qiu
Feichan
F
Shen
Yan
Y
Wang
Heng
H
Yuan
Jiangang
J
Qiang
Boqin
B
Peng
Xiaozhong
X
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000235:genetics; D008780:Methyltransferases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D020365:RNA Helicases; D012367:RNA, Viral / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology* / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D008780:Methyltransferases; C504965:Nsp13 protein, SARS-CoV; D020365:RNA Helicases