An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12.
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).
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Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
Breed
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Diederichs
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Welte
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Coulton
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D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D002614:Chelating Agents; D002846:Chromatography, Affinity; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005291:Ferrichrome; D006639:Histidine / Q000737:chemistry; D007501:Iron; D009532:Nickel; D011487:Protein Conformation; D015202:Protein Engineering; D011991:Receptors, Virus / Q000737:chemistry* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D001425:Bacterial Outer Membrane Proteins; D002614:Chelating Agents; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D011991:Receptors, Virus; D011994:Recombinant Proteins; D005291:Ferrichrome; D006639:Histidine; D009532:Nickel; D007501:Iron