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Sci Rep. 2013 Oct 4;3:2854. doi: 10.1038/srep02854.

A new protein-protein interaction sensor based on tripartite split-GFP association.

Author information

1
INSERM UMR1037, Cancer Research Center of Toulouse, Université de Toulouse, Institut Claudius Regaud, F-31052 Toulouse, France.

Abstract

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.

PMID:
24092409
PMCID:
PMC3790201
DOI:
10.1038/srep02854
[Indexed for MEDLINE]
Free PMC Article

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