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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Gene Order
  • Genetic Vectors
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • Humans
  • Molecular Imaging
  • Mutation
  • Protein Binding
  • Protein Engineering
  • Protein Interaction Mapping* / methods
  • Protein Multimerization
  • Protein Structure, Secondary
  • Recombinant Proteins*
  • Reproducibility of Results
  • Solubility
  • Tacrolimus Binding Proteins / chemistry
  • Tacrolimus Binding Proteins / genetics
  • Tacrolimus Binding Proteins / metabolism

Substances

  • Recombinant Proteins
  • Green Fluorescent Proteins
  • Tacrolimus Binding Proteins