Immunoaffinity purification of epitope-tagged human beta 2-adrenergic receptor to homogeneity.

Journal Article

To obtain large quantities of pure human beta 2-adrenergic receptor (beta 2-AR) needed for structural studies, an efficient method for beta 2-AR purification was developed using a recombinant receptor with an eight amino acid epitope at its C-terminus. This epitope is recognized by KT3-monoclonal antibody. The epitope tagged beta 2-AR was expressed in Sf9 cells with a specific activity of 5-20 pmol/mg of membrane protein. The epitope-tagged and wild-type receptors had identical ligand binding properties. The tagged receptor was solubilized using dodecyl-beta-maltoside with a quantitative yield. Solubilized epitope-tagged receptors were partially purified by KT3-mAb immunoaffinity in 60-70% yield. Further purification of the receptors on an alprenolol-affinity column resulted in a homogenous preparation with an overall yield of > 30%. The purified receptor was concentrated to > 1 mg/ml without loss of ligand binding activity.

Full Text

Duke Authors

Cited Authors

  • Kwatra, MM; Schreurs, J; Schwinn, DA; Innis, MA; Caron, MG; Lefkowitz, RJ

Published Date

  • December 1995

Published In

Volume / Issue

  • 6 / 6

Start / End Page

  • 717 - 721

PubMed ID

  • 8746622

International Standard Serial Number (ISSN)

  • 1046-5928

Digital Object Identifier (DOI)

  • 10.1006/prep.1995.0001

Language

  • eng

Conference Location

  • United States