Prokaryotic expression, in vitro folding, and molecular pharmacological characterization of the neuropeptide Y receptor type 2.
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 Y(2) 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 Y(2) receptors.
Schmidt
Peter
P
Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstrasse 16-18, D-04107 Leipzig, Germany.
Lindner
Diana
D
Montag
Cindy
C
Berndt
Sandra
S
Beck-Sickinger
Annette G
AG
Rudolph
Rainer
R
Huster
Daniel
D
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009478:Neuropeptide Y / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017476:Receptors, Neuropeptide Y / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D008823:Micelles; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y