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A NEW APPROACH FOR INSULIN RECEPTOR LIGAND SCREENING |
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Principal Investigator: T. ISSAD
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TECHNICAL DESCRIPTION
This invention involves a procedure based on Bioluminescence Resonance Energy Transfer (BRET) to monitor the activation state of the insulin receptor in vitro. A human insulin receptor with one -beta-subunit fused to Renilla luciferase (Rluc) and the other -beta-subunit fused to the Enhanced Yellow Fluorescent Protein (EYFP) has been developed. The conformational change induced by insulin and other ligands can be detected as an energy transfer (BRET signal) between Rluc and EYFP. BRET signal parallels the effects of the ligands on receptor tyrosine kinase activity. Purified chimeric insulin receptors can be prepared on a large scale and stored at -80 °C for subsequent use. This preparation can be distributed automatically in 96 well microplates and the effect of molecules on insulin receptor activity can be measured in a few minutes with the BRET methodology.
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