A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications
A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications
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DOI：
10.1038/nbt0102-87
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发表时间：
2002-01-01
影响因子：
46.9
通讯作者：
Miyawaki, A
中科院分区：
文献类型：
--
作者：
Nagai, T;Ibata, K;Miyawaki, A
水母中的绿色荧光蛋白(GFP)为生物系统提供了大量的应用。在过去的几年中，突变研究已经改善了GFP的折叠性能(参考文献)。然而，缓慢的成熟仍然是使用绿色荧光蛋白变体进行可视化的一大障碍。当GFP变体在37℃下表达和/或针对某些细胞器时，这些问题会加剧。因此，获得更有效地成熟的GFP变体对于扩大研究应用的发展至关重要。作为绿色荧光蛋白的变体，黄色荧光蛋白(YFP)对酸相对敏感，并唯一地被氯离子(Cl−)猝灭。为了使YFP具有完全和稳定的荧光，需要同时降低对pH和Cl-−敏感性的突变。在这里，我们描述了名为“金星”的YFP改进版本的开发。金星包含一种新的突变，F46L，它在37℃时极大地加速发色团的氧化，这是成熟的限速步骤。由于其他突变的结果，F64L/M153T/V163A/S175G，Venus折叠良好，对酸中毒和Cl−的暴露相对耐受。我们成功地将神经肽Y-Venus融合蛋白有效地靶向到PC12细胞的致密核颗粒。它的分泌很容易通过测量释放到介质中的荧光来监测。使用金星作为受体，可以早期检测到可靠的荧光共振能量转移(FRET)信号，用于脑片中的钙测量。随着成熟速度和效率的提高，以及对环境抵抗力的增强，金星将实现以前不可能实现的荧光标记。
The green fluorescent protein (GFP) from the jellyfishAequorea victoriahas provided a myriad of applications for biological systems. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs ,). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37°C and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. AmongAequoreaGFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl−). For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl−are desired. Here we describe the development of an improved version of YFP named “Venus”. Venus contains a novel mutation, F46L, which at 37°C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl−. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before.