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Pomerantsev A.P.,National Institute of Allergy and Infectious Diseases | Pomerantseva O.M.,Biological Defense Research Directorate | Moayeri M.,National Institute of Allergy and Infectious Diseases | Fattah R.,National Institute of Allergy and Infectious Diseases | And 2 more authors.
Protein Expression and Purification | Year: 2011

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 10 mg pure protein per liter of culture. © 2011 Elsevier Inc. All rights reserved. Source


Arolas J.L.,Proteolysis Laboratory | Botelho T.O.,Proteolysis Laboratory | Vilcinskas A.,Justus Liebig University | Gomis-Ruth F.X.,Proteolysis Laboratory
Angewandte Chemie - International Edition | Year: 2011

It goes both ways: An unprecedented mechanism of metalloendopeptidase inhibition has been identified for the insect metalloproteinase inhibitor, which is both cleaved and rejoined at bond Asn56-Ile57 by thermolysin under appropriate conditions. A two-product complex is formed after hydrolysis and, simultaneously, a Michaelis complex is poised for synthesis of a peptide bond (see crystal structure). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Arolas J.L.,Proteolysis Laboratory | Garcia-Castellanos R.,Proteolysis Laboratory | Goulas T.,Proteolysis Laboratory | Akiyama Y.,Kyoto University | Gomis-Ruth F.X.,Proteolysis Laboratory
Protein Expression and Purification | Year: 2014

Little is known about the catalytic mechanism of integral membrane (IM) peptidases. HtpX is an IM metallopeptidase that plays a central role in protein quality control by preventing the accumulation of misfolded proteins in the membrane. Here we report the recombinant overexpression and purification of a catalytically ablated form of HtpX from Escherichia coli. Several E. coli strains, expression vectors, detergents, and purification strategies were tested to achieve maximum yields of pure and well-folded protein. HtpX was successfully overexpressed in E. coli BL21(DE3) cells using a pET-derived vector attaching a C-terminal His8-tag, extracted from the membranes using octyl-β-d-glucoside, and purified to homogeneity in the presence of this detergent in three consecutive steps: cobalt-affinity, anion-exchange, and size-exclusion chromatography. The production of HtpX in milligram amounts paves the way for structural studies, which will be essential to understand the catalytic mechanism of this IM peptidase and related family members. © 2014 Elsevier Inc. All rights reserved. Source

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