Protein Expression and Purification, Vol.41, No.2, 235-240, 2005
Molecular cloning, expression, and purification of SARS-CoV nsp 13
The SARS-nsp13 protein was identified as an mRNA cap I methyltransferase. In this Study, the nsp 13 gene was cloned front 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 Eseherichia coli BL21 (DE3). The His-tag-fused protein was expressed by induction of 0.5 mM IPTG and purified by a single Ni2+ 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. (c) 2005 Elsevier Inc. All rialits reserved.