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

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