Abstract
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.
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (31971193; 31670773).
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Liu, YL., Guo, ZY. (2022). The NanoBiT-Based Homogenous Ligand–Receptor Binding Assay. In: Kim, SB. (eds) Bioluminescence. Methods in Molecular Biology, vol 2525. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2473-9_10
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DOI: https://doi.org/10.1007/978-1-0716-2473-9_10
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