The NanoBiT-Based Homogenous Ligand-Receptor Binding Assay.

NanoLuc Binary Technology (NanoBiT) was recently developed by Promega, based on a large NanoLuc fragment (LgBiT) and two small complementation tags, the low-affinity SmBiT tag and the high-affinity HiBiT tag. In recent studies, we applied NanoBiT to ligand-binding assays of some G protein-coupled receptors via genetic fusion of a secretory LgBiT (sLgBiT) to the extracellular N-terminus of the receptors and covalent attachment of the low-affinity SmBiT tag to an appropriate position of their peptide ligands. The NanoBiT-based homogenous ligand-receptor binding assay is convenient for use and suitable for both the wild-type and mutant receptors, representing a novel tool for interaction mechanism studies of these receptors with their ligands. In the present chapter, we provide detailed protocols for setting up the NanoBiT-based homogenous binding assay using growth hormone secretagogue receptor type 1a (GHSR1a) and its endogenous agonist and antagonist as a representative model system.