Automated large-scale purification of a G protein-coupled receptor for neurotensin.
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.
White
Jim F
JF
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892-8030, USA.
Trinh
Loc B
LB
Shiloach
Joseph
J
Grisshammer
Reinhard
R
D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000378:metabolism; D005285:Fermentation; D009496:Neurotensin / Q000378:metabolism*; D018028:Receptors, Neurotensin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D018028:Receptors, Neurotensin; D011993:Recombinant Fusion Proteins; C114036:neurotensin type 1 receptor; D009496:Neurotensin; D010450:Endopeptidases; C099488:TEV protease