SPR imaging-based monitoring of caspase-3 activation

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Title
SPR imaging-based monitoring of caspase-3 activation
Author(s)
Kyoungsook Park; Jun Hyoung Ahn; So Yeon Yi; Moonil Kim; Bong Hyun Chung
Bibliographic Citation
Biochemical and Biophysical Research Communications, vol. 368, no. 3, pp. 684-689
Publication Year
2008
Abstract
The activation of caspase-3 plays an important role in the apoptotic process. In this study, we describe a novel method by which caspase-3-dependent proteolytic cleavage can be monitored, using a surface plasmon resonance (SPR) imaging protein chip system. To the best of our knowledge, this is the first report regarding the SPR imaging-based monitoring of caspase-3 activation. In order to evaluate the performance of this protocol, we constructed 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 cleavage of the artificial caspase-3 substrate in response to caspase-3 using an SPR imaging sensor. The purified GST:DEVD:EGFP protein was initially immobilized onto a glutathionylated gold chip surface, and subsequently analyzed using an SPR imaging system. As a result, caspase-3 activation predicated on the proteolytic properties inherent to substrate specificity could be monitored via an SPR imaging system with a detection performance similar to that achievable by the conventional method, including fluorometric assays. Collectively, our data showed that SPR imaging protein chip system can be effectively utilized to monitor the proteolytic cleavage in caspase-3, thereby potentially enabling the detection of other intracellular protease activation via the alteration of the protease recognition site in the linker peptides.
Keyword
linker peptidecaspase-3 activationprotein chipSPR imaging system
ISSN
0006-291X
Publisher
Elsevier
DOI
http://dx.doi.org/10.1016/j.bbrc.2008.01.137
Type
Article
Appears in Collections:
Critical Diseases Diagnostics Convergence Research Center > 1. Journal Articles
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