A high-throughput method for development of FRET-based indicators for proteolysis

被引：77

作者：

Nagai, T

Miyawaki, A

机构：

[1] RIKEN, Brain Sci Inst, Adv Technol Dev Grp, Lab Cell Funct & Dynam, Wako, Saitama 3510198, Japan

[2] PRESTO, JST, Kawaguchi, Saitama 3320012, Japan

来源：

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS | 2004年 / 319卷 / 01期

基金：

日本科学技术振兴机构;

关键词：

fluorescence resonance energy transfer; green fluorescent protein; high-throughput screening; imaging; apoptosis; protease;

D O I：

10.1016/j.bbrc.2004.04.147

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：72 / 77

页数：6

共 9 条

[1]

Lakowicz J.R., 2004, PRINCIPLES FLUORESCE, V3rd

[2] Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein [J].