Novel bioluminescent receptor-binding assays for peptide hormones: using ghrelin as a model.

摘要: Peptide hormones perform important biological functions by binding specific cell membrane receptors. For hormone–receptor interaction studies, receptor-binding assays are widely used. However, conventional rely on radioactive tracers that have drawbacks. In recent we established novel non-radioactive for some recombinant protein based the ultrasensitive bioluminescence of a newly developed nanoluciferase (NanoLuc) reporter. present work, extended bioluminescent assay to peptide small size and can be conveniently prepared chemical synthesis. Human ghrelin, 28-amino acid hormone carrying special O-fatty modification, was used as model. To prepare ghrelin tracer, chemically synthesized analog with unique cysteine residue at C-terminus site-specifically conjugated an engineered NanoLuc exposed via reversible disulfide linkage. The NanoLuc-conjugated retained high affinity receptor GHSR1a (Kd = 1.14 ± 0.13 nM, n 3) able sensitively monitor various ligands. will facilitate studies its receptor. We also proposed general procedures convenient conjugation other assays.