A novel ultrasensitive bioluminescent receptor-binding assay of INSL3 through chemical conjugation with nanoluciferase |
| |
Authors: | Lei Zhang Ge Song Ting Xu Qing-Ping Wu Xiao-Xia Shao Ya-Li Liu Zeng-Guang Xu Zhan-Yun Guo |
| |
Affiliation: | 1. Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China;2. Central Laboratory, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200150, China |
| |
Abstract: | Insulin-like peptide 3 (INSL3) is a reproduction-related peptide hormone belonging to the insulin/relaxin superfamily, which mediates testicular descent in the male fetus, suppresses male germ cell apoptosis and promotes oocyte maturation in adults by activating the relaxin family peptide receptor 2 (RXFP2). To establish an ultrasensitive receptor-binding assay for INSL3−RXFP2 interaction studies, in the present work we labeled a recombinant INSL3 peptide with a newly developed nanoluciferase (NanoLuc) reporter through a convenient chemical conjugation approach, including the introduction of an active disulfide bond to INSL3 by chemical modification and engineering of a 6× His-Cys-NanoLuc carrying a unique exposed cysteine at the N-terminus. The bioluminescent NanoLuc-conjugated INSL3 retained high binding affinity with the target receptor RXFP2 (Kd = 2.0 ± 0.1 nM, n = 3) and was able to sensitively monitor the receptor-binding of a variety of ligands, representing a novel ultrasensitive tracer for non-radioactive receptor-binding assays. Our present chemical conjugation approach could readily be adapted for conjugation of NanoLuc with other proteins, even other macrobiomolecules, for various highly sensitive bioluminescent assays. |
| |
Keywords: | INSL3 Receptor-binding Chemical conjugation Bioluminescent assay Nanoluciferase |
本文献已被 ScienceDirect 等数据库收录! |
|