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Production of Biologically Active Bacillus anthracis Edema Factor in Escherichia coli
Author(s) -
Cooksey Bridget A.,
Sampey Gavin C.,
Pierre Jennifer L.,
Zhang Xiaozhen,
Karwoski Jeffrey D.,
Choi Gil H.,
Laird Michael W.
Publication year - 2004
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp049725n
Subject(s) - bacillus anthracis , anthrax toxin , escherichia coli , periplasmic space , cytosol , microbiology and biotechnology , biochemistry , biological activity , cytoplasm , biology , calmodulin , bacteria , chemistry , recombinant dna , enzyme , in vitro , fusion protein , gene , genetics
Anthrax is caused by the Gram‐positive spore‐forming bacterium Bacillus anthracis . The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA facilitates the translocation of LF and EF into the cytosol of mammalian cells. LF is thought to be a zinc‐dependent metalloprotease that results in death. EF is a calmodulin‐ and calcium‐dependent adenylate cyclase that causes edema upon entrance into the cytosol by elevating the cAMP levels in cells. Previous efforts to produce recombinant EF (rEF) in Escherichia coli yielded only 2.5 mg of rEF per liter of culture. In this work, we produced soluble rEF in large quantities in both the periplasm and cytoplasm of E. coli from shake flasks and fermentors. The rEF protein was purified by standard chromatography and yielded >97% pure, biologically active rEF. Yields of purified rEF from medium cell density fermentations resulted in up to 2.38 g/L of highly pure, biologically active rEF protein. These results exhibit the ability to generate gram quantities of active rEF from E. coli .