Anthrax Research Today is a free monthly online journal that collates and summarizes the latest research about Anthrax, including details on bacillus anthracis, contagiousness, exposure, effects. | ||||||||
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Single-step purification of recombinant anthrax lethal factor from periplasm of Escherichia coli.Chang HH, Tsai MF, Chung CP, Chen PK, Hu HI, Kau JH, Huang HH, Lin HC, Sun DS Institute of Human Genetics, Tzu-Chi University, No. 701, Section 3, Chung-Yang Road, Hualien 970, Taiwan, ROC; Institute of Molecular and Cellular Biology, Tzu-Chi University, Hualien, Taiwan, ROC; Institute of Medical Science, Tzu-Chi University, Hualien, Taiwan, ROC. 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. Published 18 October 2006 in J Biotechnol, 126(3): 277-285.
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