Biotechnol. Appl. Biochem. (2009) 52, 107–112 - J. Lu and others - Expression and purification of protective antigen

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Biotechnology and Applied Biochemistry (2009) 52, (107–112) (Printed in Great Britain)

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

Jinbiao Lu*, Dong Wei†, Yefu Wang*¹ and Guozhi Wang†¹

*State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, People's Republic of China, and †Bacterial Vaccine Department, National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, People's Republic of China

Key words: anthrax, Bacillus anthracis, Escherichia coli strain Rosetta™ 2(DE3), hydrophobic-interaction chromatography (HIC), protective antigen (PA), single-step purification.

Abbreviations used: OF, oedema factor; HIC, hydrophobic-interaction chromatography; HRP, horseradish peroxidase; IPTG, isopropyl β-D-thiogalactoside; LB, Luria–Bertani; LF, lethal factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; PA, protective antigen; rLF, recombinant LF; rPA, recombinant PA.

¹Correspondence may be addressed to either of these authors (email wangyefu@hotmail.com or tbtestlab@163.net).

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.