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. Published by Elsevier Inc.
Species">Bacillus anthracis produces a number of extracellular proteases that impaspan>ct the integrity and yield of other proteins in the span>n class="Species">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. Published by Elsevier Inc.
Authors: Gregor Grass; Angelika Schierhorn; Eduard Sorkau; Helmut Müller; Peter Rücknagel; Dietrich H Nies; Beate Fricke Journal: Infect Immun Date: 2004-01 Impact factor: 3.441
Authors: Timothy D Read; Scott N Peterson; Nicolas Tourasse; Les W Baillie; Ian T Paulsen; Karen E Nelson; Hervé Tettelin; Derrick E Fouts; Jonathan A Eisen; Steven R Gill; Erik K Holtzapple; Ole Andreas Okstad; Erlendur Helgason; Jennifer Rilstone; Martin Wu; James F Kolonay; Maureen J Beanan; Robert J Dodson; Lauren M Brinkac; Michelle Gwinn; Robert T DeBoy; Ramana Madpu; Sean C Daugherty; A Scott Durkin; Daniel H Haft; William C Nelson; Jeremy D Peterson; Mihai Pop; Hoda M Khouri; Diana Radune; Jonathan L Benton; Yasmin Mahamoud; Lingxia Jiang; Ioana R Hance; Janice F Weidman; Kristi J Berry; Roger D Plaut; Alex M Wolf; Kisha L Watkins; William C Nierman; Alyson Hazen; Robin Cline; Caroline Redmond; Joanne E Thwaite; Owen White; Steven L Salzberg; Brendan Thomason; Arthur M Friedlander; Theresa M Koehler; Philip C Hanna; Anne-Brit Kolstø; Claire M Fraser Journal: Nature Date: 2003-05-01 Impact factor: 49.962
Authors: Joseph A Bellanti; Feng-Ying C Lin; Chiayung Chu; Joseph Shiloach; Stephen H Leppla; German A Benavides; Arthur Karpas; Mahtab Moayeri; Chunyan Guo; John B Robbins; Rachel Schneerson Journal: Clin Vaccine Immunol Date: 2011-12-21
Authors: Shihui Liu; Jie Liu; Qian Ma; Liu Cao; Rasem J Fattah; Zuxi Yu; Thomas H Bugge; Toren Finkel; Stephen H Leppla Journal: Proc Natl Acad Sci U S A Date: 2016-06-29 Impact factor: 11.205
Authors: Sergey M Bezrukov; Xian Liu; Vladimir A Karginov; Alexander N Wein; Stephen H Leppla; Michel R Popoff; Holger Barth; Ekaterina M Nestorovich Journal: Biophys J Date: 2012-09-19 Impact factor: 4.033
Authors: Austen Terwilliger; Michelle C Swick; Kathryn J Pflughoeft; Andrei Pomerantsev; C Rick Lyons; Theresa M Koehler; Anthony Maresso Journal: J Bacteriol Date: 2015-05-11 Impact factor: 3.490
Authors: Joan L Arolas; Theodoros Goulas; Andrei P Pomerantsev; Stephen H Leppla; F Xavier Gomis-Rüth Journal: Structure Date: 2016-01-05 Impact factor: 5.006