Journal
FEBS LETTERS
Volume 564, Issue 3, Pages 289-293Publisher
WILEY
DOI: 10.1016/S0014-5793(04)00195-4
Keywords
large-scale purification; G protein-coupled receptor; automation; detergent; neurotensin receptor
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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 200-1 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. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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