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Biochim Biophys Acta. 2016 Feb;1863(2):284-92. doi: 10.1016/j.bbamcr.2015.11.031. Epub 2015 Nov 28.

Multiplex detection of protein-protein interactions using a next generation luciferase reporter.

Author information

1
Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia, Via Adamello 16, 20139 Milano, Italy.
2
Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, United States.
3
Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia, Via Adamello 16, 20139 Milano, Italy. Electronic address: mark.wade@iit.it.

Abstract

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 same sample, without the requirement for complex filters or deconvolution. We provide chemical and genetic validation of this system using MDM2-targeted small molecules and mutagenesis, respectively. Combined with the superior signal:noise and smaller size of split NanoLuc, this multiplexed PPI assay format can be exploited to study the induction or disruption of pairwise interactions that are prominent in many cell signaling pathways.

KEYWORDS:

Cancer; Interaction; Luciferase; MDM2; PPI; p53

PMID:
26646257
DOI:
10.1016/j.bbamcr.2015.11.031
[Indexed for MEDLINE]
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