A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins

Wednesday, October 26, 2011 — Poster Session IV
2:00 p.m. – 4:00 p.m.	Natcher Conference Center	NIAID	VIROL/MICRO-19

Authors

• AP Pomerantsev
• OM Pomerantseva
• M Moayeri
• R Fattah
• C Tallant
• SH Leppla

Abstract

Bacillus anthracis produces proteases that impact the integrity and yield of the B. anthracis secretome. Here we show that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen, lethal factor, and edema factor (EF), produced from the B. anthracis, are completely degraded at the onset of stationary phase due to the action of proteases. An improved knockout system was used to sequentially disrupt six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). The role of each protease in the degradation was demonstrated. Levels of the anthrax toxin components in the supernatant of the sporulation defective, pXO1containing strain deleted for the six proteases were significantly increased and remained stable over 24 hours of growth. A pXO1-free variant of this six-protease mutant, designated BH460, provides an improved host strain for the preparation of recombinant proteins. BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or Escherichia coli hosts. BH460 is recommended as an effective host strain for recombinant protein production, typically yielding greater than 10 mg pure protein per liter of culture.

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