Soluble expression and purification of the anthrax protective antigen in E. coli and identification of a novel dominant-negative mutant N435C |
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Authors: | Gaobing Wu Chunfang Feng Yuzhi Hong Aizhen Guo Sha Cao Junli Dong Ling Lin Ziduo Liu |
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Institution: | (1) State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China;(2) College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China; |
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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°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. |
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