Expression and purification of SARS coronavirus membrane protein.
To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The recombinants were transformed into Escherichia coli (E. Coli) BL21 (DE3) and induced by Isopropyl-beta-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0.992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.
Dai
Wuxing
W
Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430040, China.
Lei
Mingjun
M
Wu
Shaoting
S
Chen
Zhihao
Z
Liang
Liang
L
Pan
Huirong
H
Qin
Li
L
Gao
Shitong
S
Yuan
Shishan
S
Zhang
Renli
R
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry / Q000235:genetics*; D014765:Viral Vaccines / Q000096:biosynthesis
D008565:Membrane Proteins; D011994:Recombinant Proteins; D014765:Viral Vaccines