title: Assessment of a fully active class A G protein-coupled receptor isolated from in vitro folding.
abstract: We provide a protocol for the preparation of fully active Y2 G protein-coupled receptors (GPCRs). Although a valuable target for pharmaceutical research, information about the structure and dynamics of these molecules remains limited due to the difficulty in obtaining sufficient amounts of homogeneous and fully active receptors for in vitro studies. Recombinant expression of GPCRs as inclusion bodies provides the highest protein yields at lowest costs. But this strategy can only successfully be applied if the subsequent in vitro folding results in a high yield of active receptors and if this fraction can be isolated from the nonactive receptors in a homogeneous form. Here, we followed that strategy to provide large quantities of the human neuropeptide Y receptor type 2 and determined the folding yield before and after ligand affinity chromatography using a radioligand binding assay. Directly after folding, we achieved a proportion of ~25% active receptor. This value could be increased to ~96% using ligand affinity chromatography. Thus, a very homogeneous sample of the Y2 receptor could be prepared that exhibited a K(D) value of 0.1 ± 0.05 nM for the binding of polypeptide Y, which represents one of the natural ligands of the Y2 receptor.
authors: Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, D-04107 Leipzig, Germany.
authors: Mathias
authors: M
authors: Bosse
authors: Lars
authors: L
authors: Thomas
authors: Rayk
authors: R
authors: Hassert
authors: Annette G
authors: AG
authors: Beck-Sickinger
authors: Daniel
authors: D
authors: Huster
authors: Peter
authors: P
authors: Schmidt
mesh_terms: D002846:Chromatography, Affinity; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008958:Models, Molecular; D009478:Neuropeptide Y / Q000378:metabolism; D019894:Peptide YY / Q000378:metabolism; D017510:Protein Folding; D011869:Radioligand Assay; D017476:Receptors, Neuropeptide Y / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D008024:Ligands; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; D011994:Recombinant Proteins; C095889:neuropeptide Y2 receptor; D019894:Peptide YY