Development of a fluorescent three‐hybrid system for the identification of protein‐protein associators,Peptide Science

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Development of a fluorescent three‐hybrid system for the identification of protein‐protein associators
Peptide Science ( IF 1.5 ) Pub Date : 2020-06-05 , DOI: 10.1002/pep2.24178
Andrew D. Foster ₁ , Chun‐wa Chung ₂ , Michael M. Hann ₂ , Graham L. Simpson ₂ , Ali Tavassoli ₁

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The chemically‐induced dimerization of proteins is fundamental to many key regulatory pathways. A number of natural products exert their downstream effect through the stabilization of a protein complex, either by direct binding to two distinct protein partners or via an allosteric mechanism. Here, we report a bacterial three‐hybrid system, with dual life/death and fluorescent reporters, which detects protein association and is compatible with high‐throughput screening. We use rapamycin mediated mTOR‐FKBP12 (mammalian target of rapamycin—FK506 Binding Protein 12 kDa) dimerization to validate this platform; the addition of rapamycin results in the association of these target proteins, leading to the expression of two essential life/death reporter genes and a fluorescent signal. We further used this system to quantify the activity of rapamycin by utilizing the fluorescent readout, exemplifying its potential in screening and for ranking large in vivo libraries for compounds that upregulate the association of any two given proteins.

中文翻译：

荧光三杂交系统的开发，用于鉴定蛋白质-蛋白质缔合体

化学诱导的蛋白质二聚化是许多关键调控途径的基础。许多天然产物通过直接结合两个不同的蛋白质伴侣或通过变构机制来稳定蛋白质复合物，从而发挥下游作用。在这里，我们报告了一个具有生命/死亡和荧光双重报道基因的细菌三杂交系统，该系统可检测蛋白质缔合并与高通量筛选兼容。我们使用雷帕霉素介导的mTOR‐FKBP12（雷帕霉素的哺乳动物靶标-FK506结合蛋白12 kDa）二聚化来验证该平台。雷帕霉素的添加导致这些靶蛋白的缔合，导致两个必需的生命/死亡报告基因和荧光信号的表达。体内文库，用于上调任何两个给定蛋白质的缔合的化合物。

更新日期：2020-06-05

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