title: Efficient production and characterization of Bacillus anthracis lethal factor and a novel inactive mutant rLFm-Y236F.
abstract: Lethal factor (LF) is a 90kDa 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 0.2mM IPTG induction at 28 degrees 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 5mg 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.
authors.affiliation: National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Sha
authors.initials: S
authors.lastname: Cao
authors.forename: Ziduo
authors.initials: Z
authors.lastname: Liu
authors.forename: Aizhen
authors.initials: A
authors.lastname: Guo
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Chengxian
authors.initials: C
authors.lastname: Zhang
authors.forename: Wu
authors.initials: W
authors.lastname: Gaobing
authors.forename: Feng
authors.initials: F
authors.lastname: Chunfang
authors.forename: Yadi
authors.initials: Y
authors.lastname: Tan
authors.forename: Huanchun
authors.initials: H
authors.lastname: Chen
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D001408:Bacillus anthracis / Q000737:chemistry; D001427:Bacterial Toxins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D008666:Metalloendopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D051379:Mice
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin; D008666:Metalloendopeptidases