Immunoaffinity purification of epitope-tagged human beta 2-adrenergic receptor to homogeneity.
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.
Kwatra
M M
MM
Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Schreurs
J
J
Schwinn
D A
DA
Innis
M A
MA
Caron
M G
MG
Lefkowitz
R J
RJ
D000526:Alprenolol; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D000939:Epitopes / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012995:Solubility; D018411:Spodoptera
D000911:Antibodies, Monoclonal; D000939:Epitopes; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; D000526:Alprenolol