Prokaryotic Expression, In Vitro Folding, and Molecular Pharmacological Characterization of the Neuropeptide Y Receptor Type 2NPeter SchmidtDInstitute of Medical Physics and Biophysics, University of Leipzig, HaitelstraBe 16-18, D-04107 Leipzig, Germany

摘要

G protein-coupled receptors (GPCRs) are a class of membrane proteins that represent a major target for pharmacological developments. However, there is still little knowledge about GPCR structure and dynamics since high-level expression and characterization of active GPCRs in vitro is extremely complicated. Here, we describe the recombinant expression and functional folding of the human Y2 receptor from inclusion bodies of E. coli cultures. Milligram protein quantities were produced using high density fermentation and isolated in a single step purification with a yield of over 20 mg/L culture. Extensive studies were carried out on in vitro refolding and stabilization of the isolated receptor in detergent solution. The specific binding of the ligand, the 36 residue neuropeptide Y (NPY), to the recombinant Y2 receptors in micellar form was shown by several radioligand affinity assays. In competition experiments, an IC_(50) value in low nanomolar range could be determined. Further, a K_D value of 1.9 nM was determined from a saturation assay, where NPY was titrated to the recombinant Y2 receptors.