RESUMEN
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.
Asunto(s)
Cromatografía de Afinidad/métodos , Liposomas/metabolismo , Receptores Adrenérgicos alfa 2/aislamiento & purificación , Receptores Adrenérgicos alfa 2/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 2 , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Western Blotting , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Etilmaleimida/farmacología , Humanos , Ligandos , Liposomas/química , Datos de Secuencia Molecular , Fentolamina/metabolismo , Fosfatidilcolinas/metabolismo , Unión Proteica , Pliegue de Proteína , Receptores Adrenérgicos alfa 2/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Solubilidad , Tiocianatos/farmacología , Yohimbina/metabolismoRESUMEN
The objective is to generate milligram quantities of recombinant human alpha 2C2 adrenergic receptor for X-ray crystallographic studies. It has been cloned in Saccharomyces cerevisiae, and the production level is at best about 13 pmol/mg of membrane protein, as estimated by radio-ligand binding assay. The receptor is solubilized with sucrose monolaurate followed by immunoaffinity purification and reconstitution into phospholipid vesicles. The efficiency of solubilization and immuno-purification are 60% and 91%, respectively.
Asunto(s)
Receptores Adrenérgicos alfa 2/biosíntesis , Proteínas Recombinantes/biosíntesis , Humanos , Biología Molecular/economía , Receptores Adrenérgicos alfa 2/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Monoclonal antibodies (Mabs) against human alpha2C2-adrenergic receptor (alpha2C2-AR) were raised in mice and characterized. Bacterially expressed fusion protein consisting a sequence from the putative third intracellular loop (amino acids 213-343) of human alpha2C2 and glutathione-S-transferase (GST) was used as antigen. Results from mass spectrometry of purified thrombin cleaved alpha2C2 polypeptide suggested that the epitope region would lie near the aminoterminal end of the 3rd intracellular loop of human alpha2C2-AR. Elevation of Mabs was detected with Western blotting from mouse blood samples. Three alpha2C2 specific cell clones were expanded to in vitro production in hollow fiber systems. The specificity of the Mabs was further determined by immunoprecipitation and immunocytochemistry. Scatchard analysis of thrombin digested, purified, Europium-labelled antigen (amino acids 213-343 of alpha2C2) revealed binding affinity constants of 0.4 x 10(9), 0.7 x 10(9) and 1.6 x 10(9) M(-1) and Kds of 2.6, 1.4 and 0.6 nM for the three Mabs 2B1, 3G3 and 7G1, respectively.