期刊
BIOCHIMIE
卷 95, 期 12, 页码 2454-2459出版社
ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER
DOI: 10.1016/j.biochi.2013.09.008
关键词
INSL3; Receptor-binding; Chemical conjugation; Bioluminescent assay; Nanoluciferase
资金
- National Natural Science Foundation of China [31270824, 30970609]
- National Basic Research Program of China (973 Program) [2010CB912604]
- Fundamental Research Funds for the Central Universities
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 6x 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 (K-d = 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. (C) 2013 Elsevier Masson SAS. All rights reserved.
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