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

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1998

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Ferguson, Andrew D.
Breed, Jason
Coulton, James W.

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Published

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Protein Science. 1998, 7(7), pp. 1636-1638. ISSN 0961-8368. eISSN 1469-896X. Available under: doi: 10.1002/pro.5560070719

Zusammenfassung

FhuA (M, 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 quanties 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 Å resolution. The crystals belong to space group P61 or P65 with unit cell dimensions of a = b = 174 Å, c = 88 Å (α = β = 90°, γ = 120").

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Fachgebiet (DDC)
570 Biowissenschaften, Biologie

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crystallization, FhuA, membrane protein, outer membrane, protein crystals, siderophore, TonB-dependent receptor, X-ray crystallography

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ISO 690FERGUSON, Andrew D., Jason BREED, Kay DIEDERICHS, Wolfram WELTE, James W. COULTON, 1998. An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12. In: Protein Science. 1998, 7(7), pp. 1636-1638. ISSN 0961-8368. eISSN 1469-896X. Available under: doi: 10.1002/pro.5560070719
BibTex
@article{Ferguson1998inter-6877,
  year={1998},
  doi={10.1002/pro.5560070719},
  title={An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12},
  number={7},
  volume={7},
  issn={0961-8368},
  journal={Protein Science},
  pages={1636--1638},
  author={Ferguson, Andrew D. and Breed, Jason and Diederichs, Kay and Welte, Wolfram and Coulton, James W.}
}
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