An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12

Protein Sci. 1998 Jul;7(7):1636-8. doi: 10.1002/pro.5560070719.

Abstract

FhuA (Mr 78,992, 714 amino acids), siderophore receptor for ferrichrome-iron in the outer membrane of Escherichia coli, was affinity tagged, rapidly purified, and crystallized. To obtain FhuA in quantities sufficient for crystallization, a hexahistidine tag was genetically inserted into the fhuA gene after amino acid 405, which resides in a known surface-exposed loop. Recombinant FhuA405.H6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal-chelate chromatography was purified in large amounts to homogeneity. FhuA crystals were grown using the hanging drop vapor diffusion technique and were suitable for X-ray diffraction analysis. On a rotating anode X-ray source, diffraction was observed to 3.0 A resolution. The crystals belong to space group P6(1) or P6(5) with unit cell dimensions of a=b=174 A, c=88 A (alpha=beta=90 degrees, gamma=120 degrees).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Outer Membrane Proteins / chemistry*
  • Bacterial Outer Membrane Proteins / isolation & purification*
  • Chelating Agents
  • Chromatography, Affinity
  • Crystallization
  • Crystallography, X-Ray
  • Escherichia coli / chemistry*
  • Escherichia coli Proteins*
  • Ferrichrome
  • Histidine / chemistry
  • Iron
  • Nickel
  • Protein Conformation
  • Protein Engineering
  • Receptors, Virus / chemistry*
  • Receptors, Virus / isolation & purification*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification

Substances

  • Bacterial Outer Membrane Proteins
  • Chelating Agents
  • Escherichia coli Proteins
  • FhuA protein, E coli
  • Receptors, Virus
  • Recombinant Proteins
  • Ferrichrome
  • Histidine
  • Nickel
  • Iron