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Prokaryotic expression, in vitro folding, and molecular pharmacological characterization of the neuropeptide Y receptor type 2
Author(s) -
Schmidt Peter,
Lindner Diana,
Montag Cindy,
Berndt Sandra,
BeckSickinger Annette G.,
Rudolph Rainer,
Huster Daniel
Publication year - 2009
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.266
Subject(s) - g protein coupled receptor , recombinant dna , receptor , neuropeptide y receptor , radioligand , in vitro , biochemistry , chemistry , neuropeptide , inclusion bodies , radioligand assay , biology , microbiology and biotechnology , gene
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. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009