Improving analytical methods for protein-protein interaction through implementation of chemically inducible dimerization.
Improving analytical methods for protein-protein interaction through implementation of chemically inducible dimerization.
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DOI：
10.1038/srep27766
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发表时间：
2016-06-10
影响因子：
4.6
通讯作者：
Burow M
中科院分区：
文献类型：
--
作者：
Andersen TG;Nintemann SJ;Marek M;Halkier BA;Schulz A;Burow M
当在酵母双杂交或裂解GFP测定中研究具有互补报告标签的两种蛋白质之间的相互作用时，仍然难以区分真阴性结果和假阴性结果，并且难以比较实验之间的相互作用水平。这导致灵敏度降低，并使弱相互作用或短暂相互作用的分析难以执行。在这项工作中，我们描述了记者，可以化学诱导二聚化独立的调查相互作用，从而减轻这些问题的发展。我们将我们的报告员纳入广泛使用的分裂泛素，双分子荧光互补（BiFC）和Förster共振能量转移（FRET）为基础的方法，并研究了酵母和植物中不同的蛋白质-蛋白质相互作用。我们证明了这一概念的功能，通过分析弱相互作用的蛋白质从专门的代谢模式植物拟南芥。我们的研究结果表明，化学诱导的二聚化可以作为一个内置的控制分裂为基础的系统，很容易实现，并允许直接评估的功能。
When investigating interactions between two proteins with complementary reporter tags in yeast two-hybrid or split GFP assays, it remains troublesome to discriminate true- from false-negative results and challenging to compare the level of interaction across experiments. This leads to decreased sensitivity and renders analysis of weak or transient interactions difficult to perform. In this work, we describe the development of reporters that can be chemically induced to dimerize independently of the investigated interactions and thus alleviate these issues. We incorporated our reporters into the widely used split ubiquitin-, bimolecular fluorescence complementation (BiFC)- and Förster resonance energy transfer (FRET)- based methods and investigated different protein-protein interactions in yeast and plants. We demonstrate the functionality of this concept by the analysis of weakly interacting proteins from specialized metabolism in the model plant Arabidopsis thaliana. Our results illustrate that chemically induced dimerization can function as a built-in control for split-based systems that is easily implemented and allows for direct evaluation of functionality.