A high-throughput method for development of FRET-based indicators for proteolysis.
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.
Nagai
Takeharu
T
Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan.
Miyawaki
Atsushi
A
D017209:Apoptosis; D001426:Bacterial Proteins / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D004396:Coloring Agents / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
D001426:Bacterial Proteins; D004396:Coloring Agents; D018076:DNA, Complementary; D008164:Luminescent Proteins; D011506:Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D010450:Endopeptidases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases