A potent reporter applicable to the monitoring of caspase-3-dependent proteolytic cleavage

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A potent reporter applicable to the monitoring of caspase-3-dependent proteolytic cleavage
Kyoungsook Park; Hyo Jin Kang; Jun Hyoung Ahn; So Yeon Yi; Sang Hee Han; H J Park; Sang Jeon Chung; Bong Hyun Chung; Moonil Kim
Bibliographic Citation
Journal of Biotechnology, vol. 138, no. 1, pp. 17-23
Publication Year
In this study, we developed a chimeric caspase-3 substrate (GST:DEVD:EGFP) comprised of glutathione-S transferase (GST) and enhanced green fluorescent protein (EGFP) with a specialized linker peptide harboring the caspase-3 cleavage sequence, DEVD. Using this reporter, we assessed the proteolytic cleavage of the artificial caspase-3 substrate for caspase-3. The common feature of this approach is that the presence of the DEVD sequence between GST and EGFP allows for caspase-3-dependent cleavage after the Asp (D) residue, resulting in the elimination of EGFP from the GST:DEVD:EGFP reporter. To the best of our knowledge, this study reports the first application employing a chimeric protein substrate, with the similar accuracy level compared to the conventional methods such as fluorometric assays. As a result, using this GST:DEVD:EGFP reporter, caspase-3 activation based on proteolytic properties could be monitored via a variety of bioanalytical techniques such as immunoblot analysis, glutathione-agarose bead assay, and on-chip visualization, providing both technical and economical advantages over the extensively utilized fluorogenic peptide assay. Our results convincingly showed that this versatile reporter (GST:DEVD:EGFP) constitutes a useful system for the monitoring of caspase-3 activation, potentially enabling the monitoring of the proteolytic activities of different intra-cellular proteases via the substitution of the cleavage sequence within the same schematic construct.
Caspase-3 activationLabel-freeLinker peptideProtease activityProteolytic reporter
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Critical Diseases Diagnostics Convergence Research Center > 1. Journal Articles
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