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Functional Impact of Regulated Expression on Yeast Two-Hybrid


Traditional use of the yeast two-hybrid (Y2H) system is primarily for identifying unknown protein interactors to proteins of interest. Alternatively, Y2H can be adapted to make quantitative measurements of interaction strength, but there are some obstacles. For example, unequal expression of the proteins of interest will lead to false comparisons and using slightly toxic proteins can decrease cell viability. To alleviate this issue, we sought to control expression by implementing an inducible CUP1 promoter design using the major nuclear export factor, Nxf1, as bait. We observed that induction increased expression levels above that of the traditionally used constitutive ADH1 promoter in the pGBK vector. Furthermore, we showed that CUP1 driven expression levels are titratable and dependent on copper concentrations. Lastly, our findings suggest that we could quantify the changes in growth and therefore interaction strength using a liquid growth assay. Thus, this modified assay has the potential to allow for comparisons of protein interaction strength using the Y2H system.

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