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

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• 作者：Qing-Ping Wu ; Lei Zhang ; Xiao-Xia Shao ; Jia-Hui Wang ; Yu Gao ; Zeng-Guang Xu…
• 关键词：Bioluminescence ; Binding ; Ligand ; Receptor ; Relaxin ; RXFP1
• 刊名：Amino Acids
• 出版年：2016
• 出版时间：April 2016
• 年：2016
• 卷：48
• 期：4
• 页码：1099-1107
• 全文大小：1,347 KB
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• 作者单位：Qing-Ping Wu (1)
  Lei Zhang (1)
  Xiao-Xia Shao (1)
  Jia-Hui Wang (1)
  Yu Gao (1)
  Zeng-Guang Xu (1)
  Ya-Li Liu (1)
  Zhan-Yun Guo (1)
  
  1. Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China
  
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Biochemistry
  Analytical Chemistry
  Biochemical Engineering
  Life Sciences
  Proteomics
  Neurobiology
  
• 出版者：Springer Wien
• ISSN：1438-2199

文摘

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