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High‐level expression and single‐step purification of recombinant Bacillus anthracis protective antigen from Escherichia coli
Author(s) -
Lu Jinbiao,
Wei Dong,
Wang Yefu,
Wang Guozhi
Publication year - 2009
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20070245
Subject(s) - immunogen , escherichia coli , recombinant dna , ammonium sulfate , inclusion bodies , ammonium sulfate precipitation , bacillus anthracis , urea , antigen , chemistry , microbiology and biotechnology , fusion protein , chromatography , sepharose , biology , bacteria , biochemistry , gene , enzyme , size exclusion chromatography , monoclonal antibody , antibody , genetics , immunology
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.