Expression and Preparation of a G-Protein-Coupled Cannabinoid Receptor CB2 for NMR Structural Studies

Abstract

Cannabinoid receptor type II, or CB₂, is an integral membrane protein that belongs to a large class of G-protein-coupled receptors (GPCR)s. CB₂ is a part of the endocannabinoid system, which plays an important role in the regulation of immune response, inflammation, and pain. Information about the structure and function of CB₂ is essential for the development of specific ligands targeting this receptor. We present here a methodology for recombinant expression of CB₂ and its stable isotope labeling, purification, and reconstitution into liposomes, in preparation for its characterization by nuclear magnetic resonance (NMR). Correctly folded, functional CB₂ labeled with [¹³C,¹⁵N]tryptophan or uniformly labeled with ¹³C and ¹⁵N is expressed in a medium of defined composition, under controlled aeration, pH, and temperature conditions. The receptor is purified by affinity chromatography and reconstituted into lipid bilayers in the form of proteoliposomes suitable for analysis by NMR spectroscopy. © 2019 by John Wiley & Sons, Inc.