Designing Caspase‐3 Sensors for Imaging of Apoptosis in Living Cells
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A sensitive and specific caspase-3 sensor, based on a single enhanced green fluorescent protein (EGFP) to avoid the cross-talk from the overlap of cyan fluorescent protein (CFP) excitation or yellow fluorescent protein (YFP) emission spectra as observed in most FRET probes, was reported. A ratiometric protease sensor was designed by grafting a caspase-3-specific cleavage linker at a sensitive loop location (Glu172) relative to the chromophore, taking advantage of the EGFP's high resistance to proteases. To obtain optimal signal change and kinetic properties, two helical sequences were used to extend the caspase-3 cleavage linker and to improve enzymatic accessibility in solution. The comparison between dual cleavage sequence (DEVD) and a single sequence in caspase-3 sensors was also conducted. Moreover, to achieve enzymatic specificity, a recognition sequence (VDEVDG) with preferable residues from P5 to P1 was inserted into EGFP to create another sensor denoted as EGFP-C3B.
author list (cited authors)
Chen, N., Huang, Y., Yang, L., Liu, R., & Yang, J. J.