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High-Throughput Protein-Protein Interaction Assays Using Tripartite Split-GFP Complementation.

Jean-Denis Pedelacq1, Geoffrey S Waldo2, Stéphanie Cabantous3

  • 1Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, Toulouse, France.

Methods in Molecular Biology (Clifton, N.J.)
|July 4, 2019
PubMed
Summary
This summary is machine-generated.

This study introduces a split-green fluorescent protein (GFP) system to monitor protein-protein interactions in E. coli. The reconstituted GFP fluorescence intensity correlates with the strength of these crucial cellular interactions.

Keywords:
Protein fragment complementationProtein taggingProtein–protein interactionSplit-GFP

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Cellular processes rely on intricate protein-protein interaction networks.
  • Understanding these interactions is vital for deciphering cellular response mechanisms.
  • Key players and their interactions need characterization at cellular and molecular levels.

Purpose of the Study:

  • To present a protocol for monitoring protein-protein interactions.
  • To utilize a tripartite split-green fluorescent protein (GFP) system.
  • To enable interaction studies in E. coli cells and cell extracts.

Main Methods:

  • Employs a split-GFP system with GFP1-9, GFP10, and GFP11 fragments.
  • Bait and prey proteins are fused to GFP10 and GFP11.
  • Interaction reconstitutes GFP fluorescence, detectable in E. coli.

Main Results:

  • The system successfully monitors protein-protein interactions.
  • Fluorescence intensity is directly correlated with interaction strength.
  • The protocol is applicable in both intact cells and cell extracts.

Conclusions:

  • The tripartite split-GFP system provides a robust method for studying protein-protein interactions.
  • This technique aids in understanding the molecular basis of cellular functions.
  • It offers a quantitative approach to assess interaction dynamics.