PMID:9684898
| Citation |
Ferguson, AD, Breed, J, Diederichs, K, Welte, W and Coulton, JW (1998) An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12. Protein Sci. 7:1636-8 |
|---|---|
| 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). |
| Links |
PubMed PMC2144053 Online version:10.1002/pro.5560070719 |
| Keywords |
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 |
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