A Split-Luciferase Reporter Recognizing GFP and mCherry Tags to Facilitate Studies of Protein-Protein Interactions

Int J Mol Sci. 2017 Dec 11;18(12):2681. doi: 10.3390/ijms18122681.

Abstract

The use of fluorescently-tagged proteins in microscopy has become routine, and anti-GFP (Green fluorescent protein) affinity matrices are increasingly used in proteomics protocols. However, some protein-protein interactions assays, such as protein complementation assays (PCA), require recloning of each protein as a fusion with the different parts of the complementation system. Here we describe a generic system where the complementation is separated from the proteins and can be directly used with fluorescently-tagged proteins. By using nanobodies and performing tests in cell-free expression systems, we accelerated the development of multiple reporters, detecting heterodimers and homodimers or oligomers tagged with GFP or mCherry. We demonstrate that the system can detect interactions at a broad range of concentrations, from low nanomolar up to micromolar.

Keywords: Leishmania tarentolae cell-free; protein–protein interaction; split-luciferase; universal reporter.

MeSH terms

  • Cell-Free System / metabolism
  • Genes, Reporter*
  • Genetic Engineering
  • Green Fluorescent Proteins / metabolism*
  • Luciferases / genetics*
  • Luciferases / metabolism
  • Luminescent Proteins / metabolism*
  • Microscopy, Fluorescence
  • Protein Interaction Maps
  • Proteomics
  • Red Fluorescent Protein

Substances

  • Luminescent Proteins
  • Green Fluorescent Proteins
  • Luciferases