Immunoaffinity purification and reconstitution of human alpha(2)-adrenergic receptor subtype C2 into phospholipid vesicles
| Autor: | Adrian Goldman, Sari Liitti, Tuomo Glumoff, Mika Scheinin, Marja-Terttu Matikainen |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Protein Folding
Adrenergic receptor medicine.drug_class Saccharomyces cerevisiae Blotting Western Molecular Sequence Data Biology Monoclonal antibody Ligands Chromatography Affinity Phentolamine Receptors Adrenergic alpha-2 Radioligand medicine Humans Amino Acid Sequence Receptor Chromatography Elution Antibodies Monoclonal Yohimbine Adrenergic alpha-2 Receptor Antagonists biology.organism_classification Recombinant Proteins Chaotropic agent Biochemistry Solubility Ethylmaleimide Liposomes Chromatography Gel Phosphatidylcholines Electrophoresis Polyacrylamide Gel Thiocyanates Biotechnology medicine.drug Protein Binding |
| Zdroj: | Protein expression and purification. 22(1) |
| ISSN: | 1046-5928 |
| Popis: | Large quantities of correctly folded, pure α 2 -adrenergic receptor protein are needed for structural analysis. We report here the first efficient method to purify human α 2 -adrenergic receptor subtype C2 to homogeneity from recombinant yeast Saccharomyces cerevisiae by one-step purification using a monoclonal antibody column (specific for α 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. |
| Databáze: | OpenAIRE |
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