Efficient production and characterization of Bacillus anthracis lethal factor and a novel inactive mutant rLFm-Y236F
- 作者：Sha Cao ; Ziduo Liu ; Aizhen Guo ; Yan Li ; Chengxian Zhang ; Wu Gaobing ; Feng Chunfang ; Yadi Tan ; Huanchun Chen
- 关键词：Bacillus anthracis ; Lethal factor ; Anthrax ; Lethal toxin ; Protein purification
- 刊名：Protein Expression and Purification
- 出版年：2008
- 期刊代码：184_10465928
- 类别：bio
- 出版时间：May 2008
- 卷：59
- 期：1
- 页码：25-30
- 文件大小：247 K
摘要
Lethal factor (LF) is a 90 kDa zinc metalloprotease that plays an important role in the virulence of anthrax. Recombinant LF (rLF) is an effective tool to study anthrax pathogenesis and treatment. In this study, the LF gene was cloned into the Escherichia coli expression vector pGEX-6P-1 and expressed as a GST fusion protein (GST-rLF) in E. coli BL21-codonPlus (DE3)-RIL cells with 0.2 mM IPTG induction at 28 °C. The GST-rLF protein was purified and the GST-tag was then cleaved in a single step by combining both GST-affinity column and treatment with 3C protease. This procedure yielded 5 mg of rLF protein per liter of culture. The purified rLF was functional as confirmed by cytotoxicity assay in RAW264.7 cells and Western blot assay. Furthermore, the rLF could induce strong immune response in BALB/c mice and the presence of a specific antiserum could neutralize the cytotoxicity of rLF in vitro. In addition, a novel inactive mutant (rLFm-Y236F) was obtained. Compared to the wild-type rLF, an increase by 3700 folds of the purified rLFm-Y236F was needed to achieve a similar level of cytotoxicity of the wild-type rLF. This mutant might be of significance in the study of anthrax pathogenesis and treatment.