Production and purification of Bacillus anthracis protective antigen from Escherichia coli

A recombinant 63-kDa form of Bacillus anthracis protective antigen produced in the yeast Saccharomyces cerevisiae provides protection in rabbit and primate inhalational challenge models of anthrax infection

“…The only modification observed by MALDI-MS was acetylation of the N-terminal serine residue, a co-translational modification that commonly occurs in recombinantly expressed proteins [70]. The yield of rPA63 through the purification process was approximately 2.5 g of rPA63/kg of cell paste (∼181 mg/L), which compares favorably with the recovery of rPA83 from E. coli [34,35], and from a nonsporogenic, avirulent strain of B. anthracis [71].…”

“…Containment issues related to using a live, attenuated strain of B. anthracis as a host may limit consideration of this system for large-scale production of PA, but recombinant production of toxin components appears to be a safe and viable alternative [8,9,30]. Production of rPA83 in Escherichia coli has been investigated extensively [30][31][32][33][34][35], and PA83 has also been expressed in transgenic tobacco chloroplasts [36]. An alternative expression system utilizing Bacillus subtilis, a related but non-pathogenic sporulating Bacillus spp.…”

Bacillus anthracis toxins

“…A large number of reports describe the use of T7-and T5-based vectors, some incorporating signal peptides to secrete PA and PA mutant proteins to the E. coli periplasm [108,109]. With PA, LF, and EF, the use of synthetic genes having codon usage suitable for E. coli together with optimized fermentation systems has improved yields to high levels [110]. Plasmids for expression of the toxin components are available from several sources (e.g., Addgene).…”

Analytical Control Strategy of Bacterial Subunit Vaccines

“…The subunit PA anthrax vaccine has been shown in numerous animal studies to confer protective immunity against lethal aerosol challenge Williamson et al 2005;Laird et al 2004). A variety of adjuvants have been shown to be effective for inducing protective immunity of PA formulations in animal spore challenge studies, including aluminum salts, Saponin Monophsophoryl lipid A, and spore antigens (Ivins et al 1998;Berthold et al 2005;Cote et al 2012;Peachman et al 2012).…”

“…Substantial effort was given to reducing fragments observed as a major source of impurities (Rhie et al 2005;Jendrek et al 2003;Ramirez et al 2002;Miller et al 1998). Other popular derivations express and produce rPA in E. coli (Brown et al 2010;Laird et al 2004;Gwinn et al 2006;Williamson et al 2005), as pioneered by demonstration of unusually pure rPA protein from inclusion bodies (Gupta et al 1999). In one of these processes, soluble rPA is expressed in relatively high amounts in the periplasm of E. coli from shake flasks and bioreactors and purified using Q-Sepharose-HP and hydroxyapatite chromatography, and routinely found to be 96-98 % pure.…”