title: A BRET-based homogeneous insulin assay using interacting domains in the primary binding site of the insulin receptor.
abstract: A new homogeneous insulin assay requiring no chemical modification of an insulin recognition domain, which can be applied to continuous monitoring of the time-dependent cellular response in vitro, was developed. The carboxy-terminal α-chain (αCT) segment and first leucine-rich-repeat (L1) domain in the primary binding site on the insulin receptor were genetically fused with a bioluminescent protein (Nanoluc, Nluc) and a fluorescent protein (yellow fluorescent protein, YPet) to produce the insulin-sensing probe proteins Nluc-αCT and L1-YPet. The BRET signal was observed on simple mixing of insulin with these protein probes, in a so-called homogeneous assay. The BRET signal was proportional to the insulin concentration, and the lower detection limit was 0.8 μM. Time-dependent insulin secretion from drug-stimulated MIN6 cells was also successfully monitored continuously with the probe proteins. This BRET-based homogeneous insulin assay method is thus expected to be applicable to drug development by high-throughput screening.
authors: Institute for Sustainable Sciences and Development, Hiroshima University , Higashihiroshima, Hiroshima 739-8511, Japan.
authors: Hajime
authors: H
authors: Shigeto
authors: Takeshi
authors: T
authors: Ikeda
authors: Akio
authors: A
authors: Kuroda
authors: Hisakage
authors: H
authors: Funabashi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004735:Energy Transfer; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000032:analysis* / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007328:Insulin; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D011972:Receptor, Insulin; D005947:Glucose