A high-throughput method for development of FRET-based indicators for proteolysis
A high-throughput method for development of FRET-based indicators for proteolysis
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DOI：
10.1016/j.bbrc.2004.04.147
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发表时间：
2004-06-18
影响因子：
3.1
通讯作者：
Miyawaki, A
中科院分区：
文献类型：
--
作者：
Nagai, T;Miyawaki, A
SCAT 3是一种基于荧光共振能量转移（FRET）的半胱天冬酶-3活性指示剂，由增强型青色荧光蛋白、半胱天冬酶-3敏感性连接体和具有高效成熟特性的增强型黄色荧光蛋白（Venus）组成。尽管其相当大的承诺，然而，更大的响应性的荧光蛋白水解已被期望更好地理解的时空模式的caspase-3的激活过程中细胞凋亡。在本研究中，SCAT 3的连接区的长度已被彻底优化，通过使用PCR技术。使用我们自制的荧光图像分析仪筛选表达构建体的细菌菌落的高FRET效率。在哺乳动物细胞的凋亡事件中，改进的SCAT 3的FRET信号改变了约10倍，从而能够以比以前更好的空间分辨率可视化胱天蛋白酶-3活化。这种新的高通量方法将适用于开发和改进的FRET为基础的蛋白质水解的指标。(C)2004年爱思唯尔公司All rights reserved.
SCAT3 is a fluorescence resonance energy transfer (FRET)-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3-sensitive linker, and an enhanced yellow fluorescent protein with efficient maturation property (Venus). Despite its considerable promise, however, greater responsivity of fluorescence to the proteolysis has been desired for better understanding of spatio-temporal pattern of the activation of caspase-3 during apoptosis. In the present study, the length of linker regions of SCAT3 has been thoroughly optimized by use of a PCR technique. The bacterial colonies expressing the constructs were screened for high FRET efficiency using our home-made fluorescence image analyzer. The FRET signal of an improved SCAT3 changed by about tenfold during apoptotic events in mammalian cells, enabling visualization of caspase-3 activation with better spatial resolution than before. This new high-throughput method will be applicable to development and improvement of FRET-based indicators for proteolysis. (C) 2004 Elsevier Inc. All rights reserved.