Soluble expression and purification of the anthrax protective antigen in E. coli and identification of a novel dominant-negative mutant N435C.
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.
Wu
Gaobing
G
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People's Republic of China.
Feng
Chunfang
C
Hong
Yuzhi
Y
Guo
Aizhen
A
Cao
Sha
S
Dong
Junli
J
Lin
Ling
L
Liu
Ziduo
Z
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
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin