Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions
Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions
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DOI：
10.2144/000112036
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发表时间：
2006-01-01
期刊：
影响因子：
2.7
通讯作者：
Hu, CD
中科院分区：
文献类型：
--
作者：
Shyu, YJ;Liu, H;Hu, CD
蛋白质-蛋白质相互作用在协调许多细胞过程中起着关键作用。确定相互作用蛋白质的亚细胞定位和活细胞中动态相互作用的可视化对于阐明蛋白质的细胞功能至关重要。使用荧光蛋白，我们以前开发了一种双分子荧光互补（BiFC）测定和一种双分子BiFC测定，以可视化活细胞中的蛋白质-蛋白质相互作用。然而，增强型黄色荧光蛋白（YFP）的发色团成熟对较高温度的敏感性需要在观察BiFC信号之前在较低温度下预孵育。这可能会限制它们在许多信号分子研究中的应用。在这里，我们报告了新的荧光蛋白片段的识别来自金星和Cerulean的BiFC和BiFC检测在生理培养条件下。更重要的是，新鉴定的组合表现出比最初鉴定的源自YFP的片段高13倍的BiFC效率。此外，新组合的使用减少了转染所需的质粒量并缩短了孵育时间，导致特异性BiFC信号增加2倍。这些新鉴定的荧光蛋白片段将促进在生理条件下活细胞和整个动物中蛋白质-蛋白质相互作用的研究。
Protein-protein interactions play a pivotal role in coordinating many cellular processes. Determination of subcellular localization of interacting proteins and visualization of dynamic interactions in living cells are crucial to elucidate cellular functions of proteins. Using fluorescent proteins, we previously developed a bimolecular fluorescence complementation (BiFC) assay and a multicolor BiFC assay to visualize protein-protein interactions in living cells. However the sensitivity of chromophore maturation of enhanced yellow fluorescent protein (YFP) to higher temperatures requires preincubation at lower temperatures prior to visualizing the BiFC signal. This could potentially limit their applications for the study of many signaling molecules. Here we report the identification of new fluorescent protein fragments derived from Venus and Cerulean for BiFC and multicolor BiFC assays under physiological culture conditions. More importantly, the newly identified combinations exhibit a 13-fold higher BiFC efficiency than originally identified fragments derived from YFP. Furthermore, the use of new combinations reduces the amount of plasmid required for transfection and shortens the incubation time, leading to a 2-fold increase in specific BiFC signals. These newly identified fluorescent protein fragments will facilitate the study of protein-protein interactions in living cells and whole animals under physiological conditions.