Binary 2in1 Vectors Improve in Planta (Co)localization and Dynamic Protein Interaction Studies
Binary 2in1 Vectors Improve in Planta (Co)localization and Dynamic Protein Interaction Studies
复制标题
DOI：
10.1104/pp.15.00533
复制
发表时间：
2015-07-01
期刊：
影响因子：
7.4
通讯作者：
Grefen, Christopher
中科院分区：
文献类型：
--
作者：
Hecker, Andreas;Wallmeroth, Niklas;Grefen, Christopher
蛋白质-蛋白质相互作用技术是从机制层面理解体内细胞功能的重要工具。然而，只有在高效的（共）转化和积累的条件下，才能发挥其潜力，如福斯特共振能量转移（FRET）技术提供高精度和定量的相互作用数据。作为一种基于荧光的方法，FRET结合了几个优点，例如在体内环境中测量，实时背景，以及包括瞬时相互作用以及检测蛋白质的简单接近的能力。在这里，我们介绍了一种新的载体集，它结合了重组为基础的2in1克隆系统的好处，最新的最先进的荧光蛋白的最佳共积累和FRET输出研究。我们证明了它的效用在一系列的方法。将2in1克隆系统与新一代FRET荧光团对合并可以增强检测，加快克隆的制备，并使共定位研究和有意义的蛋白质-蛋白质相互作用的体内鉴定成为可能。
Fluorescence-based protein-protein interaction techniques are vital tools for understanding in vivo cellular functions on a mechanistic level. However, only under the condition of highly efficient (co)transformation and accumulation can techniques such as Forster resonance energy transfer (FRET) realize their potential for providing highly accurate and quantitative interaction data. FRET as a fluorescence-based method unifies several advantages, such as measuring in an in vivo environment, real-time context, and the ability to include transient interactions as well as detecting the mere proximity of proteins. Here, we introduce a novel vector set that incorporates the benefit of the recombination-based 2in1 cloning system with the latest state-of-the-art fluorescent proteins for optimal coaccumulation and FRET output studies. We demonstrate its utility across a range of methods. Merging the 2in1 cloning systemwith new-generation FRET fluorophore pairs allows for enhanced detection, speeds up the preparation of clones, and enables colocalization studies and the identification of meaningful protein-protein interactions in vivo.