Multiplex detection of protein–protein interactions using a next generation luciferase reporter

Publication date: February 2016 Source:Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Volume 1863, Issue 2 Author(s): Lisette G.G.C. Verhoef, Michela Mattioli, Fernanda Ricci, Yao-Cheng Li, Mark Wade Cell-based assays of protein–protein interactions (PPIs) using split reporter proteins can be used to identify PPI agonists and antagonists. Generally, such assays measure one PPI at a time, and thus counterscreens for on-target activity must be run in parallel or at a subsequent stage; this increases both the cost and time during screening. Split luciferase systems offer advantages over those that use split fluorescent proteins (FPs). This is since split luciferase offers a greater signal:noise ratio and, unlike split FPs, the PPI can be reversed upon small molecule treatment. While multiplexed PPI assays using luciferase have been reported, they suffer from low signal:noise and require fairly complex spectral deconvolution during analysis. Furthermore, the luciferase enzymes used are large, which limits the range of PPIs that can be interrogated due to steric hindrance from the split luciferase fragments. Here, we report a multiplexed PPI assay based on split luciferases from Photinus pyralis (firefly luciferase, FLUC) and the deep-sea shrimp, Oplophorus gracilirostris (NanoLuc, NLUC). Specifically, we show that the binding of the p53 tumor suppressor to its two major negative regulators, MDM2 and MDM4, can be simultaneously measured within the s...
Source: Biochimica et Biophysica Acta (BBA) Molecular Cell Research - Category: Molecular Biology Source Type: research