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Automated large‐scale purification of a G protein‐coupled receptor for neurotensin
Author(s) -
White Jim F,
Trinh Loc B,
Shiloach Joseph,
Grisshammer Reinhard
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(04)00195-4
Subject(s) - neurotensin , neurotensin receptor , receptor , chromatography , affinity chromatography , fusion protein , protein purification , escherichia coli , chemistry , g protein coupled receptor , tandem affinity purification , tobacco etch virus , protease , biochemistry , biology , neuropeptide , virus , recombinant dna , enzyme , gene , plant virus , potyvirus , virology
Structure determination of integral membrane proteins requires milligram amounts of purified, functional protein on a regular basis. Here, we describe a protocol for the purification of a G protein‐coupled neurotensin receptor fusion protein at the 3‐mg or 10‐mg level using immobilized metal affinity chromatography and a neurotensin column in a fully automated mode. Fermentation at a 200‐l scale of Escherichia coli expressing functional receptors provides the material needed to feed into the purification routine. Constructs with tobacco etch virus protease recognition sites at either end of the receptor allow the isolation of neurotensin receptor devoid of its fusion partners. The presented expression and purification procedures are simple and robust, and provide the basis for crystallization experiments of receptors on a routine basis.