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 protein are needed for structural analysis. We report here the first efficient method to purify human alpha(2)-adrenergic receptor subtype C2 to homogeneity from recombinant yeast Saccharomyces cerevisiae by one-step purification using a monoclonal antibody column (specific for alpha(2)C2). We show that the adrenoceptor antagonist phentolamine stabilized the receptor during purification. We used a very effective chaotropic agent, NaSCN, to elute the receptor from the immunoaffinity column with an overall yield of 34% before reconstitution. Ligand binding of detergent-solubilized, immunoaffinity-purified receptors could not be demonstrated, but partial recovery of ligand binding activity was achieved when purified receptors were reconstituted into phospholipid vesicles. The reconstituted receptors still bound radioligand after storage on ice for 4 weeks. This purification procedure can be easily scaled-up and thus demonstrates the utility of a monoclonal antibody column and NaSCN elution to purify large quantities of G-protein-coupled receptors.
Liitti
S
S
The Centre for Biotechnology, University of Turku and Abo Akademi University, Tykistökatu 6B, Biocity, FIN-20521, Finland
Matikainen
M T
MT
Scheinin
M
M
Glumoff
T
T
Goldman
A
A
D058669:Adrenergic alpha-2 Receptor Antagonists; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010646:Phentolamine / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics; D012995:Solubility; D013861:Thiocyanates / Q000494:pharmacology; D015016:Yohimbine / Q000378:metabolism
D058669:Adrenergic alpha-2 Receptor Antagonists; D000911:Antibodies, Monoclonal; D008024:Ligands; D008081:Liposomes; D010713:Phosphatidylcholines; D018341:Receptors, Adrenergic, alpha-2; D011994:Recombinant Proteins; D013861:Thiocyanates; D015016:Yohimbine; C024553:sodium thiocyanate; D005033:Ethylmaleimide; D010646:Phentolamine