Expression and NMR Structural Studies of Isotopically Labeled Cannabinoid Receptor Type II

Cannabinoid receptor type II (CB₂) is an integral membrane protein with seven transmembrane helices that belongs to the large superfamily of rhodopsin-like G protein-coupled receptors. The CB₂ is a part of the endocannabinoid system that plays a vital role in regulation of immune response, inflammation, pain, and other metabolic processes. Information about the structure and function of CB₂ in cell membranes is essential for development of specific pharmaceuticals that target CB₂ signaling. Methodology for recombinant expression, stable isotope labeling, purification, reconstitution into liposomes, and NMR characterization of functionally active CB₂ is presented. Uniformly ¹³C-, ¹⁵N-labeled CB₂ protein is expressed by fermentation of Escherichia coli in a medium of defined composition under controlled aeration, pH, and temperature and purified by tandem affinity chromatography. The receptor reconstituted into lipid bilayers is suitable for structural studies by solid-state NMR spectroscopy.