High-level expression and single-step purification of recombinant Bacillus anthracis protective antigen from Escherichia coli.
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.
Lu
Jinbiao
J
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, People's Republic of China.
Wei
Dong
D
Wang
Yefu
Y
Wang
Guozhi
G
D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D001408:Bacillus anthracis / Q000276:immunology*; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D002460:Cell Line; D002845:Chromatography; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D015870:Gene Expression; D057927:Hydrophobic and Hydrophilic Interactions; D002479:Inclusion Bodies / Q000737:chemistry; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D013997:Time Factors
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011994:Recombinant Proteins; C030325:anthrax toxin