Monitoring of cleavage preference for caspase-3 using recombinant protein substrates.
The apoptotic caspases have been classified in accordance with their substrate specificities, as the optimal tetrapeptide recognition motifs for a variety of caspases have been determined via positional scanning substrate combinatorial library technology. Here, we focused on two proteolytic recognition motifs, DEVD and IETD, owing to their extensive use in cell death assay. Although DEVE and IETD have been generally considered to be selective for caspase-3 and -8, respectively, the proteolytic cleavage of these substrates does not display absolute specificity for a particular caspase. Thus, we attempted to monitor the cleavage preference for caspase-3, particularly using the recombinant protein substrates. For this aim, the chimeric GST:DEVD:EGFP and GST:IETD:EGFP proteins were genetically constructed by linking GST and EGFP with the linkers harboring DEVD and IETD. To our best knowledge, this work constitutes the first application for the monitoring of cleavage preference employing the recombinant protein substrates that simultaneously allow for mass and fluorescence analyses. Consequently, GST: IETD:EGFP was cleaved partially in response to caspase-3, whereas GST:DEVD:EGFP was completely proteolyzed, indicating that GST:DEVD:EGFP is a better substrate than GST:IETD:EGFP for caspase-3. Collectively, using these chimeric protein substrates, we have successfully evaluated the feasibility of the recombinant protein substrate for applicability to the monitoring of cleavage preference for caspase-3.
Park
Kyoungsook
K
BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea.
Yi
So Yeon
SY
Kim
Un-Lyoung
UL
Lee
Chang-Soo
CS
Chung
Jin Woong
JW
Chung
Sang J
SJ
Kim
Moonil
M
D001224:Aspartic Acid / Q000032:analysis; D053148:Caspase 3 / Q000737:chemistry / Q000378:metabolism*; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers; D004789:Enzyme Activation; D005784:Gene Amplification; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics*; D015151:Immunoblotting / Q000379:methods; D009842:Oligopeptides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; D009842:Oligopeptides; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D001224:Aspartic Acid; D053148:Caspase 3; D003545:Cysteine