Purification of recombinant G-protein-coupled receptors.
Structural and functional analysis of most G-protein-coupled receptors (GPCRs) requires their expression and purification in functional form. The produced amount of recombinant membrane-inserted receptors depends on the optimal combination of a particular GPCR and production host; optimization of expression is still a matter of trial-and-error. Prior to purification, receptors must be extracted from the membranes by use of detergent(s). The choice of an appropriate detergent for solubilization and purification is crucial to maintain receptors in their functional state. The initial enrichment can be carried out by affinity chromatography using a general affinity tag (e.g., poly-histidine tag). If the first purification step does not yield pure receptor protein, purification to homogeneity can often be achieved by use of a subsequent receptor-specific ligand column. If suitable immobilized ligands are not available, size exclusion chromatography or other techniques need to be applied. Many GPCRs become unstable upon detergent extraction from lipid membranes, and measures for stabilization are discussed. As an example, the purification of a functional neurotensin receptor to homogeneity in milligram quantities is given below.
Grisshammer
Reinhard
R
Department of Health and Human Services, National Institutes of Health, National Institute of Neurological Disorders and Stroke, Rockville, Maryland, USA.
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000302:isolation & purification*; D018028:Receptors, Neurotensin / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility
D043562:Receptors, G-Protein-Coupled; D018028:Receptors, Neurotensin; D011994:Recombinant Proteins; C114036:neurotensin type 1 receptor