Production of Biologically Active Bacillus anthracis Edema Factor in Escherichia coli

Abstract

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.

Bacillus anthracisEdema and Lethal Toxin Have Different Hemodynamic Effects but Function Together to Worsen Shock and Outcome in a Rat Model

“…It is very likely, however, that hypotension with ETx is, in part, related to the adenyl cyclase activity of EF [6,28]. The recombinant ETx preparation investigated stimulated adenyl cyclase in CHO-K1 cells, just as ETx prepared from live B. anthracis did previously [6,23]. In the present in vitro studies, the efficient delivery of EF to the cytosol by PA binding caused rapid increases in levels of intracellular cAMP.…”

“…Toxin components (PA, LF, and EF) were recombinant proteins prepared and purified from Escherichia coli by standard chromatographic techniques described elsewhere [22][23][24]. Endotoxin levels were !0.1 EU/mg protein for each component as based on testing with a limulus amebocyte lysate assay.…”

Bacillus anthracisEdema Toxin Acts as an Adjuvant for Mucosal Immune Responses to Nasally Administered Vaccine Antigens

“…The role of the adenylate cyclase activity for the adjuvanticity remains to be elucidated. In this regard, the EF used in our studies contains a S447N mutation that could account for its recently reported 20-fold lower ability to induce cAMP when compared with recombinant native EF protein (33). Several mutants of both CT and LT-I, which are devoid of ADP-ribosyl transferase activity, were shown to retain their mucosal adjuvant activity and the ability to promote high mucosal IgA Ab responses.…”

“…173) and was produced in a recombinant strain of B. anthracis using an expression plasmid constructed by S. H. Leppla. This EF protein contains a S447N mutation and was shown to display ϳ20-fold less enzymatic activity than the native EF (33). Controls included mice nasally immunized with OVA plus 1 g of CT (List Biological Laboratories).…”

Anthrax edema toxin has cAMP-mediated stimulatory effects and high-dose lethal toxin has depressant effects in an isolated perfused rat heart model

“…In all experiments except for experiment 7 (comparison of Sprague-Dawley and Lewis rats), toxin components (protective antigen, lethal factor, and edema factor) were recombinant proteins prepared from Escherichia coli as previously described (5,13,20). Edema toxin and lethal toxin were composed of edema or lethal factors, respectively, with protective antigen in ratios of 1:2 on the basis of weight (the molecular weights of all three components are very similar).…”