A novel ultrasensitive bioluminescent receptor-binding assay of INSL3 through chemical conjugation with nanoluciferase.
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.
Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China; Central Laboratory, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200150, China.
Lei
L
Zhang
Ge
G
Song
Ting
T
Xu
Qing-Ping
QP
Wu
Xiao-Xia
XX
Shao
Ya-Li
YL
Liu
Zeng-Guang
ZG
Xu
Zhan-Yun
ZY
Guo
D057809:HEK293 Cells; D006801:Humans; D007328:Insulin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics; D008163:Luminescent Measurements / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D011993:Recombinant Fusion Proteins
D007328:Insulin; C084797:Leydig insulin-like protein; D011506:Proteins; C472867:RXFP2 protein, human; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D008156:Luciferases
Bioluminescent assay; Chemical conjugation; INSL3; Nanoluciferase; Receptor-binding