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

Part of the Methods in Molecular Biology book series (MIMB,volume 2025)


Most cellular processes are driven by complex protein–protein interaction networks. Identifying key players and characterizing their interactions at the cellular and molecular level is of key importance to understand biochemical mechanisms that control cellular responses. Here, we detail a protocol for monitoring protein–protein interactions in E. coli cells or in cell extracts using a tripartite split-GFP system comprised of a protein interaction detector fragment (GFP ß-strands 1 through 9 or GFP1–9) and small tagging fragments of GFP ß-strands 10 (GFP10) and 11 (GFP11). Interaction of bait and prey proteins fused to GFP10 and GFP11 tether the small GFP fragments, allowing self-association with GFP1–9. In this scenario, fluorescence intensity of the reconstituted GFP is correlated with the strength of interaction.

Key words

  • Split-GFP
  • Protein–protein interaction
  • Protein tagging
  • Protein fragment complementation

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  • DOI: 10.1007/978-1-4939-9624-7_20
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Correspondence to Stéphanie Cabantous .

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Pedelacq, JD., Waldo, G.S., Cabantous, S. (2019). High-Throughput Protein–Protein Interaction Assays Using Tripartite Split-GFP Complementation. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY.

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9623-0

  • Online ISBN: 978-1-4939-9624-7

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