Rapid preparation of bioluminescent tracers for relaxin family peptides using sortase-catalysed ligation

摘要: Relaxin family is a group of peptide hormones with variety biological functions by activating G protein-coupled receptors RXFP1–4. We recently developed bioluminescent tracers for their receptor-binding assays chemical conjugation the ultrasensitive NanoLuc reporter. To simplify preparation tracers, in present study, we established sortase-catalysed ligation approach using chimeric R3/I5 as model. Following catalysis recombinant sortase A, reporter carrying LPETG recognition motif at C-terminus was efficiently ligated to an four successive Gly residues A-chain N-terminus, via formation bond between C-terminal LPET sequence and N-terminal residue R3/I5. Saturation binding demonstrated that NanoLuc-ligated retained high affinity RXFP3 RXFP4, calculated dissociation constants (K d) 4.34 ± 0.33 nM (n = 3) 5.66 ± 0.54 nM (n = 3), respectively. Using tracer competition assays, potencies various ligands towards RXFP4 were conveniently quantified. This work provides simple method rapid relaxin peptides other protein/peptide ligand–receptor interaction studies.