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Title 

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

Authors 

Kyoungsook ParkHyo Jin KangJun Hyoung AhnSo Yeon YiSang Hee HanH J ParkSang Jeon ChungBong Hyun ChungMoonil Kim

Publisher 

Elsevier

Issue Date 

2008

Citation 

Journal of Biotechnology, vol. 138, no. 1, pp. 17-23

Keywords 

caspase-3 activationlabel-freelinker peptideprotease activityproteolytic reporteractivation analysisaminesarsenicglycosylationmedical imaging

Abstract 

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.

ISSN 

0168-1656

Link 

http://dx.doi.org/10.1016/j.jbiotec.2008.07.1999

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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