FRET-sensor for imaging with lifetime resolution

FRET-sensor with nonfluorescent protein as an acceptor was synthesized to observe caspase-3 activity in lifetime mode. We inserted caspase-3 cleavable linker between red highly fluorescent protein TagRFP and chromoprotein KFP. Dynamic light scattering was used to determine size of the fusion protein. Incubation with caspase-3 lead to increase both fluorescence intensity and lifetime of the construction. Cleavage of the linker between proteins was confirmed by electrophoresis and immunoblotting. FLIM-microscopy showed the differences between fluorescence decays of A549 cell line expressed TagRFP and TagRFP-23-KFP.