A Modified Yeast Two-Hybrid Platform Enables Dynamic Control of Expression Intensities to Unmask Properties of Protein-Protein Interactions

Erez Feuer, Gil Zimran, Michal Shpilman, Assaf Mosquna*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

The yeast two-hybrid (Y2H) assay is widely used for protein-protein interaction characterization due to its simplicity and accessibility. However, it may mask changes in affinity caused by mutations or ligand activation due to signal saturation. To overcome this drawback, we modified the Y2H system to have tunable protein expression by introducing a fluorescent reporter and a pair of synthetic inducible transcription factors to regulate the expression of interacting components. We found that the application of inducers allowed us to adjust the concentrations of interacting proteins to avoid saturation and observe interactions otherwise masked in the canonical Y2H assay, such as the abscisic acid-mediated increase in affinity of monomeric abscisic acid receptors to the coreceptor. When applied in future studies, our modified system may provide a more accurate characterization of protein-protein interactions.

Original languageAmerican English
Pages (from-to)2589-2598
Number of pages10
JournalACS Synthetic Biology
Volume11
Issue number8
DOIs
StatePublished - 19 Aug 2022

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