Assessment of a Fully Active Class A G Protein-Coupled Receptor Isolated from in Vitro Folding

作者：Bosse Mathias; Thomas Lars; Hassert Rayk; Beck Sickinger Annette G; Huster Daniel; Schmidt Peter^*

来源：Biochemistry, 2011, 50(45): 9817-9825.

DOI：10.1021/bi201320e

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 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 similar to 25% active receptor. This value could be increased to similar 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.

• 出版日期2011-11-15
• 单位河北医科大学