The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, Rehovot 7610000, Israel.
ACS Synth Biol. 2022 Aug 19;11(8):2589-2598. doi: 10.1021/acssynbio.2c00192. Epub 2022 Jul 27.
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.
酵母双杂交 (Y2H) 测定法因其简单易用而被广泛用于蛋白质-蛋白质相互作用的表征。然而，由于信号饱和，它可能会掩盖突变或配体激活引起的亲和力变化。为了克服这一缺点，我们通过引入荧光报告基因和一对合成诱导转录因子来修改 Y2H 系统，以实现可调节的蛋白质表达，从而调节相互作用成分的表达。我们发现，诱导剂的应用使我们能够调整相互作用蛋白的浓度，以避免饱和，并观察到在经典 Y2H 测定法中被掩盖的相互作用，例如脱落酸介导的单体脱落酸受体与共受体亲和力的增加。在未来的研究中应用时，我们修改的系统可能会更准确地描述蛋白质-蛋白质相互作用。
