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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
Author(s) -
Ferguson Andrew D.,
Coulton James W.,
Breed Jason,
Diederichs Kay,
Welte Wolfram
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560070719
Subject(s) - escherichia coli , bacterial outer membrane , colicin , ferrichrome , crystallization , crystallography , siderophore , chemistry , integral membrane protein , membrane protein , biology , membrane , biochemistry , gene , organic chemistry
FhuA (M r 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 hexahisti‐dine tag was genetically inserted into the fhuA gene after amino acid 40.5, which resides in a known surface‐exposed loop. Recom‐binant FhuA40.5.H 6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal‐chelate chroma‐tography 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 P6 1 or P6 5 with unit cell dimensions of a = b = 174 Å, c = 88 Å (α = β = 90°,γ = 120°).