Figure 20.

Schematic representation a cyclic luciferase assay for the detection of caspase-3 activity. DnaE fragments flank the FLuc fragments, which are connected via the amino acid sequence DEVD. N- and C-termini of FLuc ligate after translation and protein splicing, forming an inactive cyclic luciferase. Caspase-3 will cut the cyclic luciferase at DEVD, activating it and creating bioluminescence. A degradation sequence d4PEST is also included in this fusion protein design, such that only cyclic forms of the biosensor are allowed to exist. DEVD: substrate sequence of caspase-3; Asp-Glu-Val-Asp. DnaE: catalytic α subunit of DNA polymerase III. PEST: sequence which is known to accelerate degradation of a fused protein. d4PEST: mutated murine PEST sequence.