title: Single-step purification of recombinant anthrax lethal factor from periplasm of Escherichia coli.
abstract: Lethal toxin (LT) that composed by protective antigen and lethal factor (LF) is the major virulence factor of Bacillus anthracis. The treatments of LT in animals could reproduce most manifestations of B. anthracis infections that greatly improves our knowledge in LT-mediated pathogenesis and facilitates anthrax-related researches without having to directly contact the hazardous bacterium B. anthracis. The recombinant protein of LF (rLF), however, still lacks a simple purification method. Herein, we developed single-step nickel affinity purification of rLF with yield up to 3mg/l. By fusion to the leader sequence of outer membrane protein OmpA, rLF could easily be purified from the periplasm of Escherichia coli. To investigate whether the rLT is functional in our system, both wild type rLF and the catalytic mutant rLF that contains a single amino acid substitution at zinc-binding site (LF(E687A)), were subjected to macrophage cytotoxicity analysis. Our data showed that the rLT is fully functional, while the LF(E687A) fail to induce cell death of tested macrophage cells. These findings suggested that the purification protocol herein is a user-friendly method that allows researchers to obtain the functional rLF by single-step purification.
authors.affiliation: Institute of Human Genetics, Tzu-Chi University, No. 701, Section 3, Chung-Yang Road, Hualien 970, Taiwan, ROC.
authors.forename: Hsin-Hou
authors.initials: HH
authors.lastname: Chang
authors.forename: Mei-Fang
authors.initials: MF
authors.lastname: Tsai
authors.forename: Chia-Pei
authors.initials: CP
authors.lastname: Chung
authors.forename: Po-Kong
authors.initials: PK
authors.lastname: Chen
authors.forename: Hsin-I
authors.initials: HI
authors.lastname: Hu
authors.forename: Jyh-Hwa
authors.initials: JH
authors.lastname: Kau
authors.forename: Hsin-Hsien
authors.initials: HH
authors.lastname: Huang
authors.forename: Hung-Chi
authors.initials: HC
authors.lastname: Lin
authors.forename: Der-Shan
authors.initials: DS
authors.lastname: Sun
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000302:isolation & purification* / Q000494:pharmacology; D001408:Bacillus anthracis / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000302:isolation & purification* / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects; D051379:Mice; D019897:Periplasm / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000302:isolation & purification; D052616:Solid Phase Extraction / Q000379:methods*
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011994:Recombinant Proteins; C030325:anthrax toxin