title: Production, recovery and immunogenicity of the protective antigen from a recombinant strain of Bacillus anthracis.
abstract: The protective antigen (PA) is one of the three components of the anthrax toxin. It is a secreted nontoxic protein with a molecular weight of 83 kDa and is the major component of the currently licensed human vaccine for anthrax. Due to limitations found in the existing vaccine formulation, it has been proposed that genetically modified PA may be more effective as a vaccine. The expression and the stability of two recombinant PA (rPA) variants, PA-SNKE-deltaFF-E308D and PA-N657A, were studied. These proteins were expressed in the nonsporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-deltaFF-E308D, which lacks two proteolysis-sensitive sites, is more stable than PA-N657A. Process development was conducted to establish an efficient production and purification process for PA-SNKE-deltaFF-E308D. pH, media composition, growth strategy and protease inhibitors composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only source of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time was 14-18 h after inoculation, and EDTA (5 mM) was added upon harvest for proteolysis control. Recovery of the rPA was performed by expanded-bed adsorption (EBA) on a hydrophobic interaction chromatography (HIC) resin, eliminating the need for centrifugation, microfiltration and diafiltration. The EBA step was followed by ion exchange and gel filtration. rPA yields before and after purification were 130 and 90 mg/l, respectively. The purified rPA, without further treatment, treated with small amounts of formalin or adsorbed on alum, induced, high levels of IgG anti-PA with neutralization activities.
authors: Biotechnology Unit, LCDB, NIDDK, National Institutes of Health (NIH), Bethesda, MD 20892, USA.
authors: D M
authors: DM
authors: Ramirez
authors: S H
authors: SH
authors: Leppla
authors: R
authors: R
authors: Schneerson
authors: J
authors: J
authors: Shiloach
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D001681:Biological Assay; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005260:Female; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D051379:Mice; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C030325:anthrax toxin