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bioRxiv · 10.1101/2021.07.01.450807

Inducible Yeast Two-Hybrid with Quantitative Measures

Hernandez, J.·Ross, K. D.·Hamilton, B. A.
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Abstract

The yeast two-hybrid (Y2H) assay has long been used to identify new protein-protein interaction pairs and to compare relative interaction strengths. Traditional Y2H formats may be limited, however, by use of constitutive strong promoters if expressed proteins have toxic effects or post-transcriptional expression differences in yeast among a comparison group. As a step toward more quantitative Y2H assays, we modified a common vector to use an inducible CUP1 promoter, which showed quantitative induction of several "bait" proteins with increasing copper concentration. Using mouse Nxf1 (homologous to yeast Mex67p) as a model bait, copper titration achieved levels that bracket levels obtained with the constitutive ADH1 promoter. Using a liquid growth assay for an auxotrophic reporter in multiwell plates allowed log-phase growth rate to be used as a measure of interaction strength. These data demonstrate the potential for quantitative comparisons of protein-protein interactions using the Y2H system.

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Hernandez, J., Ross, K. D., Hamilton, B. A.. 2021-07-02. Inducible Yeast Two-Hybrid with Quantitative Measures. https://doi.org/10.1101/2021.07.01.450807

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