title: Soluble expression and purification of the anthrax protective antigen in E. coli and identification of a novel dominant-negative mutant N435C.
abstract: The anthrax toxin is an AB-type bacterium toxin composed of the protective antigen (PA) as the cell-binding B component, and the lethal factor (LF) and edema toxin (EF) as the catalytic A components. The PA component is a key factor in anthrax-related research and recombinant PA can be produced in general in Escherichia coli. However, such recombinant PA always forms inclusion bodies in the cytoplasm of E. coli, making difficult the procedure of its purification. In this study, we found that the solubility of recombinant PA was dramatically enhanced by fusion with glutathione S-transferase (GST) and an induction of its expression at 28 degrees C. The PA was purified to high homogeneity and a yield of 3 mg protein was obtained from 1 l culture by an affinity-chromatography approach. Moreover, we expressed and purified three PA mutants, I394C, A396C, and N435C, which were impaired in expression in previous study. Among them, a novel mutant N435C which conferred dominant-negative inhibitory activity on PA was identified. This new mutant may be useful in designing new antitoxin for anthrax prophylaxis and therapy.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People's Republic of China.
authors.forename: Gaobing
authors.initials: G
authors.lastname: Wu
authors.forename: Chunfang
authors.initials: C
authors.lastname: Feng
authors.forename: Yuzhi
authors.initials: Y
authors.lastname: Hong
authors.forename: Aizhen
authors.initials: A
authors.lastname: Guo
authors.forename: Sha
authors.initials: S
authors.lastname: Cao
authors.forename: Junli
authors.initials: J
authors.lastname: Dong
authors.forename: Ling
authors.initials: L
authors.lastname: Lin
authors.forename: Ziduo
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000881:Anthrax / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D001408:Bacillus anthracis / Q000276:immunology / Q000502:physiology; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D012995:Solubility
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin