UAS regulatory sequences drive expression of 'iCasper', an infra-red fluorescent reporter of executioner-caspase activity. The core of the iCasper protein is an engineered, circularly permutated infra-red fluorescent protein (cp-IFP) in which the N and C termini have been truncated and are linked by the caspase-3/7 cleavage sequence DEVDG (an additional Gly residue is added before the cleavage sequence as a flexible linker). Flanking this core are two complementary split-GFP coding sequences. In order to fluoresce, IFPs require a biliverdin (BV) cofactor that is autocatalytically incorporated by being bound non-covalently to the IFP GAF domain and via a thioester bond to a conserved 'catalytic' Cys residue near the N-terminus of the protein. The changes in the iCasper cp-IFP core result in the BV-binding cavity of the GAF and the catalytic Cys residue being further part than in the native IFP, preventing the incorporation of the BV cofactor. Thus, in the absence of cleavage by caspase-3/7, the iCasper cp-IFP is inactive, and is not infra-red fluorescent. Upon cleavage by the caspase, the BV cofactor can be incorporated, resulting in infra-red fluorescence. The presence of the flanking split-GFP fragments ensures that the cp-IFP protein remains intact after cleavage. In addition, it means that that iCasper protein shows green fluorescence in both inactive and active states. The PBac{UAS-iCasper-T2A-HO1} construct also produces the Hsap\HMOX1 protein, which is generates the BV cofactor required by the iCasper cp-IFP. The two iCasper and Hsap\HMOX1 proteins are separated by a self-cleaving T2A motif.
This is a UAS construct that expresses two proteins.
The first protein is called iCasper and it has five parts: 1) split GFP T:Avic\GFP 2) half of an infrared fluorescent protein (IFP) (Zzzz\BrBphP) 3) a caspase cleavage site (T:Zzzz\Caspase37.CS) 4) the other IFP half 5) the other split GFP half.
The split GFP domains bind to each other and hold the two IFP domains together so that they can fluoresce when caspase cuts the peptide holding them apart.
The second protein is a human heme oxygenase (Hsap\HMOX1) needed for the IFP to fluoresce. The two proteins are separated by a self-cleaving T2A motif.