Characterization of proteinase activation dynamics by capillary electrophoresis conjugating with fluorescent protein-based probe.
In this paper, a novel strategy was reported to characterize dynamics of proteinase activation based on capillary electrophoresis (CE), using caspase-2 as the model system. A fusion protein conjugating an amino acid sequence VDVAD with two fluorescent proteins enhanced cyan fluorescence protein (ECFP) and red fluorescence protein (DsRed), ECFP-VDVAD-DsRed, was specially designed and expressed in HeLa cells as the substrate of proteinase caspase-2. In this substrate, the sequence VDVAD could be specifically recognized and cleaved by caspase-2 as soon as its activation was initiated with treatment of a certain dose of cisplatin to HeLa cells, which led to a break of the substrate into two fragments. Analyses of the cell lysates using CE in a time course of the apoptosis illustrated the dynamics of caspase-2 activation. It showed that the employment of fusion fluorescent protein greatly facilitated both CE separation and identification of the analytes. This result from cell colony by CE was compared with that from single cell achieved by optical imaging.
Zhou
Shixia
S
The Key Laboratory of Biomedical Photonics of MOE - Huibei Bioinformatics and Molecular Imaging Key Laboratory, Department of Systems Biology, College of Life Science & Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
Lin
Juqiang
J
Du
Wei
W
Zhang
Zhihong
Z
Luo
Qingming
Q
Liu
Bi-Feng
BF
Dai
Yiqun
Y
D019075:Electrophoresis, Capillary / Q000379:methods*; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D010447:Peptide Hydrolases / Q000378:metabolism*
D005456:Fluorescent Dyes; D010447:Peptide Hydrolases