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

• Published in: Amino acids 2017-09, Vol.49 (9), p.1611-1617
• Main Authors: Wang, Jia-Hui, Shao, Xiao-Xia, Hu, Meng-Jun, Wei, Dian, Nie, Wei-Han, Liu, Ya-Li, Xu, Zeng-Guang, Guo, Zhan-Yun
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Aminoacyltransferases - genetics; Aminoacyltransferases - isolation & purification; Aminoacyltransferases - metabolism; Analytical Chemistry; Assaying; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Binding; Binding, Competitive; Biochemical Engineering; Biochemistry; Biological Assay; Biomedical and Life Sciences; C-Terminus; Catalysis; Chains; Cloning, Molecular; Conjugation; Constants; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - isolation & purification; Cysteine Endopeptidases - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; G protein-coupled receptors; Gene Expression; Genes, Reporter; Genetic Vectors - chemistry; Genetic Vectors - metabolism; HEK293 Cells; Hormones; Humans; Kinetics; Life Sciences; Luciferases - genetics; Luciferases - metabolism; Luminescent Measurements - methods; N-Terminus; Nanostructure; Neurobiology; Original Article; Peptide hormones; Peptides; Proteomics; Receptors; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Receptors, Peptide - genetics; Receptors, Peptide - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Relaxin; Relaxin - genetics; Relaxin - metabolism; Sortase; Staphylococcus aureus - chemistry; Staphylococcus aureus - enzymology; Tracers
• ISSN: 0939-4451; 1438-2199
• DOI: 10.1007/s00726-017-2455-9

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.