title: Expression and purification of functional human mu opioid receptor from E.coli.
abstract: N-terminally his-tagged human mu opioid receptor, a G protein-coupled receptor was produced in E.coli employing synthetic codon-usage optimized constructs. The receptor was expressed in inclusion bodies and membrane-inserted in different E.coli strains. By optimizing the expression conditions the expression level for the membrane-integrated receptor was raised to 0.3-0.5 mg per liter of culture. Milligram quantities of receptor could be enriched by affinity chromatography from IPTG induced cultures grown at 18°C. By size exclusion chromatography the protein fraction with the fraction of alpha-helical secondary structure expected for a 7-TM receptor was isolated, by CD-spectroscopy an alpha-helical content of ca. 45% was found for protein solubilised in the detergent Fos-12. Receptor in Fos-12 micelles was shown to bind endomorphin-1 with a K(D) of 61 nM. A final yield of 0.17 mg functional protein per liter of culture was obtained.
authors: Institute of Complex Systems (ICS-6), Research Center Jülich, Jülich, Germany.
authors: Yanbin
authors: Y
authors: Ma
authors: Jan
authors: J
authors: Kubicek
authors: Jörg
authors: J
authors: Labahn
mesh_terms: D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics; D009842:Oligopeptides / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017450:Receptors, Opioid, mu / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D009842:Oligopeptides; D017450:Receptors, Opioid, mu; C105293:endomorphin 1