AN INTERNAL AFFINITY-TAG FOR PURIFICATION AND CRYSTALLIZATION OF THE SIDEROPHORE RECEPTOR FHUA, INTEGRAL OUTER-MEMBRANE PROTEIN FROM ESCHERICHIA-COLI K-12

FhuA (M-r 78,992, 714 amino acids), siderophore receptor for ferrichro me-iron in the outer membrane of Escherichia coli, was affinity tagged , rapidly purified, and crystallized. To obtain FhuA in quanties suffi cient for crystallization, a hexahistidine tag was genetically inserte d into the fhuA gene after amino acid 405, which resides in a known su rface-exposed loop. Recombinant FhuA405.H-6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal-ch elate chromatography was purified in large amounts to homogeneity. Fhu A 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 Angstrom resolution. Th e crystals belong to space group P6(1) or P6(5) with unit cell dimensi ons of a = b = 174 Angstrom, c = 88 Angstrom (alpha = beta = 90 degree s, gamma = 120 degrees).