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A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association

2013; Nature Portfolio; Volume: 3; Issue: 1 Linguagem: Inglês

10.1038/srep02854

2045-2322

Stéphanie Cabantous, Hau B. Nguyen, Jean Denis Pedelacq, Faten Koraïchi, Anu Chaudhary, Kumkum Ganguly, Meghan A. Lockard, Gilles Favre, Thomas C. Terwilliger, Geoffrey S. Waldo,

Cellular transport and secretion

Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.