title: A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins.
abstract: Bacillus anthracis produces a number of extracellular proteases that impact the integrity and yield of other proteins in the B. anthracis secretome. In this study we show that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the B. anthracis Ames 35 strain (pXO1⁺, pXO2⁻), are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). The role of each protease in degradation of the B. anthracis toxin components and ALO was demonstrated. Levels of the anthrax toxin components and ALO in the supernatant of the sporulation defective, pXO1⁺ A35HMS mutant strain deleted for the six proteases were significantly increased and remained stable over 24 h. A pXO1-free variant of this six-protease mutant strain, designated BH460, provides an improved host strain for the preparation of recombinant proteins. As an example, 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 10mg pure protein per liter of culture.
authors.affiliation: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Andrei P
authors.initials: AP
authors.lastname: Pomerantsev
authors.forename: Olga M
authors.initials: OM
authors.lastname: Pomerantseva
authors.forename: Mahtab
authors.initials: M
authors.lastname: Moayeri
authors.forename: Rasem
authors.initials: R
authors.lastname: Fattah
authors.forename: Cynthia
authors.initials: C
authors.lastname: Tallant
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Leppla
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics* / Q000378:metabolism; D001408:Bacillus anthracis / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D017353:Gene Deletion; D055786:Gene Knockout Techniques; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011994:Recombinant Proteins; C030325:anthrax toxin; D010447:Peptide Hydrolases