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

J Am Chem Soc. 2019 Mar 20;141(11):4526-4530. doi: 10.1021/jacs.8b13042. Epub 2019 Mar 6.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis*
  • Drosophila melanogaster
  • Genes, Reporter / genetics*
  • Green Fluorescent Proteins / chemistry*
  • Green Fluorescent Proteins / genetics*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Molecular Imaging*
  • Peptide Hydrolases / chemistry*
  • Peptide Hydrolases / genetics*
  • Protein Conformation, beta-Strand

Substances

  • Green Fluorescent Proteins
  • Peptide Hydrolases