Combiningin situproteolysis and mass spectrometry to crystallizeEscherichia coliPgaB Academic Article uri icon

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abstract

  • The periplasmic poly-β-1,6-N-acetyl-D-glucosamine (PNAG) de-N-acetylase PgaB fromEscherichia coliwas overexpressed and purified, but was recalcitrant to crystallization. Use of thein situproteolysis technique produced crystals of PgaB, but these crystals could not be optimized for diffraction studies. By analyzing the initial crystal hits using SDS–PAGE and mass spectrometry, the boundaries of the protein species that crystallized were determined. The re-engineered protein target crystallized reproducibly without the addition of protease and with significantly increased crystal quality. Crystals of the selenomethionine-incorporated protein exhibited the symmetry of space groupP212121and diffracted to 2.1 Å resolution.

authors

  • Little, Dustin J
  • Whitney, John
  • Robinson, Howard
  • Yip, Patrick
  • Nitz, Mark
  • Howell, P Lynne

publication date

  • July 1, 2012