title: Design, recombinant expression and convenient A-chain N-terminal europium-labelling of a fully active human relaxin-3 analogue.
abstract: Relaxin-3 (also known as INSL7) is a recently identified neuropeptide belonging to the insulin/relaxin superfamily. It plays a putative role in the regulation of food intake, in the stress response and in reproduction by activating the G-protein-coupled receptor, RXFP3. In a previous study, we prepared 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)/Eu(3+)-labelled human relaxin-3 as a tracer for the study of ligand-receptor interactions, which necessitated a complicated site-specific labelling strategy because human relaxin-3 contains four primary amine moieties, all of which react with the primary amine-specific modification reagent. To simplify the labelling procedure, in the present study we created an easily labelled, recombinant analogue of human relaxin-3 with only one primary amine moiety at the A-chain N-terminus. The analogue retained full activity and could be easily labelled by various functional probes at the A-chain N-terminus. The DOTA/Eu(3+)-labelled analogue retained high binding affinity for its cognate receptor, RXFP3, and thus represents a useful, nonradioactive and stable tracer for studying the interaction of RXFP3 with various natural or synthetic ligands. This new analogue is also a suitable template for the design of other relaxin-3 analogues that can be easily labelled with the DOTA/Eu(3+) moiety and used to study binding activity and interactions with various RXFP3 analogues in the future.
authors.affiliation: Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai, China.
authors.forename: Wei-Jie
authors.initials: WJ
authors.lastname: Zhang
authors.forename: Xiao
authors.initials: X
authors.lastname: Luo
authors.forename: Ge
authors.initials: G
authors.lastname: Song
authors.forename: Xin-Yi
authors.initials: XY
authors.lastname: Wang
authors.forename: Xiao-Xia
authors.initials: XX
authors.lastname: Shao
authors.forename: Zhan-Yun
authors.initials: ZY
authors.lastname: Guo
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D015195:Drug Design; D005063:Europium / Q000737:chemistry*; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D017510:Protein Folding; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012065:Relaxin / Q000235:genetics / Q000378:metabolism*; D012107:Research Design
chemical_list: D006573:Heterocyclic Compounds, 1-Ring; C480238:RLN3 protein, human; C410092:RXFP3 protein, human; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D005063:Europium; D012065:Relaxin