Prokaryotic expression,in vitro folding,and molecular pharmacological characterization of the neuropeptide Y receptor type 2 |
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Authors: | Peter Schmidt Diana Lindner Cindy Montag Sandra Berndt Annette G. Beck‐Sickinger Rainer Rudolph Daniel Huster |
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Affiliation: | 1. Institute of Medical Physics and Biophysics, University of Leipzig, H?rtelstra?e 16‐18, D‐04107 Leipzig, Germany;2. Institute of Biochemistry/Biotechnology, Martin Luther University Halle‐Wittenberg, D‐06120 Halle, Germany;3. Institute of Biochemistry, University of Leipzig, Brüderstra?e 34, D‐04103 Leipzig, Germany |
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Abstract: | 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 IC50 value in low nanomolar range could be determined. Further, a KD value of 1.9 nM was determined from a saturation assay, where NPY was titrated to the recombinant Y2 receptors. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 |
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Keywords: | GPCR membrane protein expression Y2 receptor E. coli in vitro folding |
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