Towards NMR structural studies on the peripheral cannabinoid receptor CB2 in micellar solution.

Human peripheral‐type cannabinoid receptor (CB2) is a G protein‐coupled membrane receptor (GPCR) involved in the immune and hormonal response. Because of its importance in signal transduction pathways, this membrane protein has become a target for drug design. Development of novel ligands would benefit greatly from NMR structural studies on ligands in the receptor‐bound state and the protein. Functional CB2 was successfully expressed in E.coli as a fusion with maltose‐binding protein (MBP), thioredoxin (TrxA) and a (His)10 tag (construct 107). The receptor was purified by Ni‐NTA affinity chromatography to about 80% purity as judged by silver staining of SDS‐PAGE gels. In order to further purify the protein, several constructs were designed which carry a second tag. The construct 109, which carries a biotin ligase recognition site (BRS) downstream of the CB2 sequence, produced in vivo biotinylated protein. However, introduction of this tag did not significantly increase the purity of the protein after elution from a monomeric avidin column. Insertion of the BRS upstream of the CB2 sequence (construct 112) led to a low level of in vivo biotinylation. This protein was successfully biotinylated in vitro and cleaved with Tev protease. Purification of the protein with a monomeric avidin column resulted in a highly enriched CB2 fraction. The efficiency of Tev protease cleavage was tested on a series of CB2‐fusions with one or two cleavage sites. Full cleavage of the fusion at mg‐quantities was achieved in micellar solution at 4oC. The cleaved CB2‐fusion in bacterial membranes retained functionality as shown by ligand binding. Construct 115, with a second decahistidine tag at the N‐terminus of CB2, enabled efficient purification of the cleaved CB2 on a Ni‐NTA column.