Immunoaffinity purification and reconstitution of human alpha(2)-adrenergic receptor subtype C2 into phospholipid vesicles

Large quantities of correctly folded, pure alpha (2)-adrenergic receptor pr otein are needed for structural analysis. We report here the first efficien t method to purify human alpha (2)-adrenergic receptor subtype C2 to homoge neity from recombinant yeast Saccharomyces cerevisiae by one-step purificat ion using a monoclonal antibody column (specific for alpha (2)C2). We show that the adrenoceptor antagonist phentolamine stabilized the receptor durin g purification. We used a very effective chaotropic agent, NaSCN, to elute the receptor from the immunoaffinity column with an overall yield of 34% be fore reconstitution. Ligand binding of detergent-solubilized, immunoaffinit y-purified receptors could not be demonstrated, but partial recovery of lig and binding activity was achieved when purified receptors were reconstitute d into phospholipid vesicles. The reconstituted receptors still bound radio ligand after storage on ice for 4 weeks. This purification procedure can be easily scaled-up and thus demonstrates the utility of a monoclonal antibod y column and NaSCN elution to purify large quantities of G-protein-coupled receptors. (C) 2001 Academic Press.