title: Expression and purification of SARS coronavirus membrane protein.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430040, China.
authors.forename: Wuxing
authors.initials: W
authors.lastname: Dai
authors.forename: Mingjun
authors.initials: M
authors.lastname: Lei
authors.forename: Shaoting
authors.initials: S
authors.lastname: Wu
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Chen
authors.forename: Liang
authors.initials: L
authors.lastname: Liang
authors.forename: Huirong
authors.initials: H
authors.lastname: Pan
authors.forename: Li
authors.initials: L
authors.lastname: Qin
authors.forename: Shitong
authors.initials: S
authors.lastname: Gao
authors.forename: Shishan
authors.initials: S
authors.lastname: Yuan
authors.forename: Renli
authors.initials: R
authors.lastname: Zhang
mesh_terms: 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
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; D014765:Viral Vaccines