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Expression of human peripheral cannabinoid receptor for structural studies
Author(s) -
Yeliseev Alexei A.,
Wong Karen K.,
Soubias Olivier,
Gawrisch Klaus
Publication year - 2005
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.051550305
Subject(s) - chemistry , biochemistry , maltose binding protein , affinity chromatography , cannabinoid receptor type 2 , cannabinoid , membrane , escherichia coli , receptor , cannabinoid receptor , chromatography , fusion protein , recombinant dna , agonist , enzyme , gene
Human peripheral‐type cannabinoid receptor (CB2) was expressed in Escherichia coli as a fusion with the maltose‐binding protein, thioredoxin, and a deca‐histidine tag. Functional activity and structural integrity of the receptor in bacterial protoplast membranes was confirmed by extensive binding studies with a variety of natural and synthetic cannabinoid ligands. E. coli membranes expressing CB2 also activated cognate G‐proteins in an in vitro coupled assay. Detergent‐solubilized receptor was purified to 80%–90% homogeneity by affinity chromatography followed by ion‐exchange chromatography. By high‐resolution NMR on the receptor in DPC micelles, it was determined that purified CB2 forms 1:1 complexes with the ligands CP‐55,940 and anandamide. The receptor was successfully reconstituted into phosphatidylcholine bilayers and the membranes were deposited into a porous substrate as tubular lipid bilayers for structural studies by NMR and scattering techniques.