A potent reporter applicable to the monitoring of caspase-3-dependent proteolytic cleavage.
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.
Park
Kyoungsook
K
BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Eoeun-Dong, Yuseong-Gu, Daejeon 305-333, Republic of Korea.
Kang
Hyo-Jin
HJ
Ahn
Junhyoung
J
Yi
So Yeon
SY
Han
Sang Hee
SH
Park
Hye-Jung
HJ
Chung
Sang J
SJ
Chung
Bong Hyun
BH
Kim
Moonil
M
D053148:Caspase 3 / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D015398:Signal Transduction / Q000235:genetics*
D049452:Green Fluorescent Proteins; D053148:Caspase 3