RRC ID 50077
Author Nagai T, Miyawaki A.
Title A high-throughput method for development of FRET-based indicators for proteolysis.
Journal Biochem. Biophys. Res. Commun.
Abstract 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.
Volume 319(1)
Pages 72-7
Published 2004-6-18
DOI 10.1016/j.bbrc.2004.04.147
PII S0006291X04008496
PMID 15158444
MeSH Apoptosis Bacterial Proteins / metabolism Caspase 3 Caspases / metabolism* Coloring Agents / pharmacology DNA, Complementary / metabolism Endopeptidases / chemistry Escherichia coli / metabolism Fluorescence Resonance Energy Transfer / methods* Green Fluorescent Proteins HeLa Cells Humans Image Processing, Computer-Assisted Luminescent Proteins / chemistry Luminescent Proteins / metabolism Microscopy, Fluorescence Polymerase Chain Reaction Proteins / chemistry* Recombinant Proteins / chemistry Time Factors
IF 2.559
Resource
DNA material SCAT3.1/pcDNA4 HMB (RDB15705).