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Protein Science (2005), 14:2638-2653. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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Expression of human peripheral cannabinoid receptor for structural studies

Alexei A. Yeliseev, Karen K. Wong, Olivier Soubias and Klaus Gawrisch

Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892, USA

(RECEIVED April 28, 2005; FINAL REVISION July 19, 2005; ACCEPTED July 19, 2005)

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.

Keywords: cannabinoid receptor; CB2; E. coli; NMR; GPCR; anodic aluminum oxide

Abbreviations: CHAPS, 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate • CHS, cholesteryl hemisuccinate Tris salt • GPCR, G-protein-coupled receptor • H10, deca-histidine tag • DM, n-dodecyl-{beta}-D-maltoside • MBP, E. coli maltose-binding protein • OG, n-octyl-{beta}-D-glucopyranoside • Tev, tobacco etch virus • TrxA, E. coli thioredoxin • SOPC, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine • POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine • DPC, dodecylphosphocholine • MOPS, 3-(N-Morpholino)propanesulfonic acid

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051550305.

Reprint requests to: Alexei Yeliseev or Klaus Gawrisch, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892, USA; e-mail: yeliseeva{at}mail.nih.gov or gawrisch{at}helix.nih.gov; fax: (301) 594-0035.


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