Spectral profiling for the simultaneous observation of four distinct fluorescent proteins and detection of protein-protein interaction via fluorescence resonance energy transfer in tobacco leaf nuclei
Spectral profiling for the simultaneous observation of four distinct fluorescent proteins and detection of protein-protein interaction via fluorescence resonance energy transfer in tobacco leaf nuclei
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DOI：
10.1104/pp.005496
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发表时间：
2002-07-01
期刊：
影响因子：
7.4
通讯作者：
Lam, E
中科院分区：
文献类型：
--
作者：
Kato, N;Pontier, D;Lam, E
控制蛋白质的亚细胞定位及其在体内与其他伙伴的相互作用是提供其功能和调控线索的重要参数。高分辨率同时跟踪多种蛋白质物种的能力将为研究和表征各种类型的翻译后控制途径提供有价值的分析系统。在这项工作中，我们建立了系统和一种涉及“光谱分析”的方法，用于使用数字成像技术在同一视场中分辨四种不同的荧光蛋白标签。利用这些技术，我们已经(a)开发了mGFP5的新衍生物，它通常用于植物领域，亮度约为三倍；(b)证实与转录因子融合的四种光谱上不同的荧光蛋白（青色、绿色、黄色和红色）可以在细胞核中稳定表达并在烟草叶肉细胞中被区分；(c)表明，可以通过测量荧光共振能量转移来检测二聚体转录因子的配对体之间的相互作用。这些技术将有助于人们有效地研究蛋白质之间的相互作用，特别是在体内条件下研究核蛋白。
The control of subcellular localization of proteins and their interaction with other partners in vivo are important parameters that provide clues to their function and regulation. The ability to simultaneously track multiple protein species with high resolution should provide a valuable assay system to study and characterize various types of posttranslational control pathways. In this work, we established the system and a method involving "spectral profiling" for the resolution of four different fluorescent protein tags in the same viewing field using digital imaging technology. With these techniques, we have (a) developed new derivatives of mGFP5, which is commonly used in the plant field, that are about three times brighter; (b) demonstrated that four spectrally distinct fluorescent proteins (cyan, green, yellow, and red) that are fused to a transcription factor could be stably expressed in nuclei and distinguished in tobacco (Nicotiana tabacum) mesophyll cells; and (c) shown that interaction between partners of a dimeric transcription factor can be detected by measuring fluorescence resonance energy transfer. These technologies should help one to study protein-protein interactions efficiently, especially for nuclear proteins under in vivo conditions.