Title of article :
Characterization of proteinase activation dynamics by capillary electrophoresis conjugating with fluorescent protein-based probe
Author/Authors :
Zhou، نويسنده , , Shixia and Lin، نويسنده , , Juqiang and Du، نويسنده , , Wei and Zhang، نويسنده , , Zhihong and Luo، نويسنده , , Qingming and Liu، نويسنده , , Bi-Feng and Dai، نويسنده , , Yiqun، نويسنده ,
Issue Information :
روزنامه با شماره پیاپی سال 2006
Abstract :
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.
Keywords :
Caspase-2 , protein activation , fluorescent protein , Dynamics , Capillary electrophoresis
Journal title :
Journal of Chromatography B
Journal title :
Journal of Chromatography B