Application of the novel bioluminescent ligand–receptor binding assay to relaxin-RXFP1 system for interaction studies

摘要: Relaxin is a prototype of the relaxin family peptide hormones and plays important biological functions by binding activating G protein-coupled receptor RXFP1. To study their interactions, in present work, we applied newly developed bioluminescent ligand-receptor assay to relaxin-RXFP1 system. First, fully active easily labeled relaxin, which three Lys residues human relaxin-2 were replaced Arg, was prepared through overexpression single-chain precursor Pichia pastoris vitro enzymatic maturation. Thereafter, B-chain N-terminus chemically cross-linked with C-terminal cysteine residue an engineered NanoLuc disulfide linkage. Receptor-binding assays demonstrated that NanoLuc-conjugated retained high affinity RXFP1 (K d = 1.11 ± 0.08 nM, n 3) able sensitively monitor variety ligands Using novel assay, highly conserved Arg relaxin-3 had distinct contributions In summary, our work provides for system facilitate interaction studies, such as characterization analogues or screening agonists antagonists