title: Expression of human peripheral cannabinoid receptor for structural studies.
abstract: 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.
authors.affiliation: Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892, USA. yeliseeva@mail.nih.gov
authors.forename: Alexei A
authors.initials: AA
authors.lastname: Yeliseev
authors.forename: Karen K
authors.initials: KK
authors.lastname: Wong
authors.forename: Olivier
authors.initials: O
authors.lastname: Soubias
authors.forename: Klaus
authors.initials: K
authors.lastname: Gawrisch
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry*; D011487:Protein Conformation; D043885:Receptor, Cannabinoid, CB2 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D043885:Receptor, Cannabinoid, CB2; D011993:Recombinant Fusion Proteins