Screening family C GPCRs for heterologous expression in baculovirus‐infected insect cells

G‐protein coupled‐receptors (GPCRs) are the largest group of membrane proteins in the human genome and are divided into six families based on genetic similarity. They are characterized by a seven alpha helix transmembrane domain and their association with heterotrimeric G‐proteins. Many GPCRs are difficult to express in cell culture and a challenge to produce in prokaryotic expression systems. Due to the difficulties with purifying membrane proteins, only a few full‐length GPCR structures have been determined, all of which belong to family A (rhodopsin‐like). Family C GPCRs differ from family A GPCRs due to the presence of a large extracellular ligand‐binding domain and include receptors activated by glutamate, GABA, calcium, pheromones, and taste molecules. We aim to overexpress and purify family C GPCRs for structural studies. We have tested a large number of family C GCPRs for expression using GFP (green fluorescent protein)‐tagged constructs and fluorescence‐based size exclusion chromatography (FSEC) and have found promising candidates that express in a dimeric homogeneous state in mammalian cells and in baculovirus‐infected insect cells. Using small‐scale (2mL) expression trials, we have used the FSEC screen to test detergents for solubilization and stabilization behavior. We are coupling expression studies with functional assays in baculovirus‐infected insect cells using calcium release assays. These experiments lay the groundwork for attempts at obtaining milligram quantities of purified class C GPCRs for structural and functional studies.