title: Molecular cloning, expression, and purification of SARS-CoV nsp13.
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.
authors.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.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Fan
authors.forename: Kunpeng
authors.initials: K
authors.lastname: Peng
authors.forename: Xinyu
authors.initials: X
authors.lastname: Tan
authors.forename: Bin
authors.initials: B
authors.lastname: Yin
authors.forename: Xiuhua
authors.initials: X
authors.lastname: Dong
authors.forename: Feichan
authors.initials: F
authors.lastname: Qiu
authors.forename: Yan
authors.initials: Y
authors.lastname: Shen
authors.forename: Heng
authors.initials: H
authors.lastname: Wang
authors.forename: Jiangang
authors.initials: J
authors.lastname: Yuan
authors.forename: Boqin
authors.initials: B
authors.lastname: Qiang
authors.forename: Xiaozhong
authors.initials: X
authors.lastname: Peng
mesh_terms: 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
chemical_list: D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D008780:Methyltransferases; C504965:Nsp13 protein, SARS-CoV; D020365:RNA Helicases