Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695, United States.
Golden LEAF Biomanufacturing Training and Education Center, North Carolina State University, Raleigh, North Carolina 27695, United States.
ACS Synth Biol. 2021 Mar 19;10(3):505-514. doi: 10.1021/acssynbio.0c00472. Epub 2021 Feb 15.
Quantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well as characterization of binding proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two-hybrid (qYY2H) system that not only enables the discovery of specific protein-protein interactions but also efficient, quantitative estimation of their binding affinities (). In qYY2H, the bait and prey proteins are expressed as yeast cell surface fusions using yeast surface display. We developed a semiempirical framework for estimating the of monovalent bait-prey interactions, using measurements of bait-prey yeast-yeast binding, which is mediated by multivalent interactions between yeast-displayed bait and prey. Using qYY2H, we identified interaction partners of SMAD3 and the tandem WW domains of YAP from a cDNA library and characterized their binding affinities. Finally, we showed that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein.
定量蛋白质-蛋白质相互作用的结合亲和力对于阐明生化信号通路中的联系以及从组合文库中分离的结合蛋白的特征至关重要。我们描述了一种定量酵母-酵母双杂交（qYY2H）系统，该系统不仅能够发现特定的蛋白质-蛋白质相互作用，而且还能够有效地、定量地估计它们的结合亲和力（）。在 qYY2H 中，诱饵和猎物蛋白被表达为酵母细胞表面融合物，使用酵母表面展示。我们开发了一种半经验框架，用于估计单价诱饵-猎物相互作用的亲和力，使用测量诱饵-猎物酵母-酵母结合，这是由酵母展示的诱饵和猎物之间的多价相互作用介导的。使用 qYY2H，我们从 cDNA 文库中鉴定了 SMAD3 和 YAP 的串联 WW 结构域的相互作用伙伴，并表征了它们的结合亲和力。最后，我们表明 qYY2H 还可以定量评估诱饵蛋白上翻译后修饰介导的结合相互作用。
