Expression of human peripheral cannabinoid receptor for structural studies.
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.
Yeliseev
Alexei A
AA
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
Wong
Karen K
KK
Soubias
Olivier
O
Gawrisch
Klaus
K
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
D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D043885:Receptor, Cannabinoid, CB2; D011993:Recombinant Fusion Proteins