High-level expression and single-step purification of recombinant Bacillus anthracis protective antigen from...

PA (protective antigen) is a major immunogen of the vaccine against anthrax. In the present study, a new expression system, Escherichia coli strain Rosetta 2(DE3), was used for high-level expression of rPA (recombinant PA) whose gene contains 66 rare E. coli codons (9.0% of 733 total PA gene codons). The rPA-formed inclusion bodies were washed with Triton X-100 and 2 M urea and solubilized in 5 M urea, followed by a 60%-satd.-ammonium sulfate precipitation. Finally, the untagged rPA was efficiently and rapidly purified by single-step hydrophobic-interaction chromatography using Phenyl-Sepharose High Performance resin on an AKTA Purifier 10 system. The yield was approx. 13 mg of high-purity (>99%) biologically active rPA per litre of culture, which can be used for the detection of anthrax and as a potential component of a vaccine against anthrax.