Designing a Green Fluorogenic Protease Reporter by Flipping a Beta Strand of GFP for Imaging Apoptosis in Animals

A family of proteases
called caspases mediate apoptosis signaling
in animals. We report a GFP-based fluorogenic protease reporter, dubbed
“FlipGFP”, by flipping a beta strand of the GFP. Upon
protease activation and cleavage, the beta strand is restored, leading
to reconstitution of the GFP and fluorescence. FlipGFP-based TEV protease
reporter achieves 100-fold fluorescence change. A FlipGFP-based executioner
caspase reporter visualized apoptosis in live zebrafish embryos with
spatiotemporal resolution. FlipGFP also visualized apoptotic cells
in the midgut of Drosophila. Thus,
the FlipGFP-based caspase reporter will be useful for monitoring apoptosis
during animal development and for designing reporters of proteases
beyond caspases. The design strategy can be further applied to a red
fluorescent protein for engineering a red fluorogenic protease reporter.