Monitoring of proteinase activation in cell apoptosis by capillary electrophoresis with bioengineered fluorescent probe

In this article, capillary electrophoresis (CE) was demonstrated as a novel means to monitor the activation of proteinase during cell apoptosis. A unique fluorescent probe that fused a specific amino acid sequence DEVD with two fluorescent proteins enhanced cyan fluorescent protein (ECFP) and red fluorescent protein (DsRed), ECFP-DEVD-DsRed, was engineered in HeLa cells as a substrate of proteinase caspase-3. Molecular imaging in vivo was conducted to evaluate the stability and reliability of this fusion protein probe expressed in HeLa cells. With treatment of a certain dose of cisplatin to HeLa cells, apoptosis was initiated, and then caspase-3 was activated that could specifically recognize the DEVD site and would subsequently cleave the constructed fluorescent probe into two pieces of protein fragments. Analyzing the cell lysates with CE in vitro at a series of time points gave a clear description of activation process of caspase-3 in cell apoptosis. Several parameters that influenced CE separation quality were optimized, such as buffer type, pH value, concentration and applied voltage. The employment of two color fluorescent proteins significantly simplified the separation and identification of the residues of the cleavage reaction.