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 Y-2 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 IC50 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 Y-2 receptors. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1732-1739, 2009