120th General Meeting of the KCS

Type Poster Presentation
Area Life Chemistry
Room No. Exhibition Hall 2+3
Time 10월 19일 (목요일) 11:00~12:30
Code BIO.P-255
Subject Wash-free labeling of target proteins and the use of photochemical handles in live cells
Authors Euiyeon Lee, Youngeun Kwon*
Department of Biomedical Engineering (BK21 plus), Dongguk University, Korea
Abstract Monitoring protein functions, interactions and movements in living cells is critical for understanding their roles in biological system. In particular, fluorescent probes allow detection of molecular interactions, mobility and conformational changes of proteins in live cells with high temporal and spatial resolution. In this study, we utilized Npu DnaE split-intein mediated protein trans-splicing (PTS) reaction for site-specific fluorescent labeling of target protein. The large N-intein (IN), is expressed using DNA recombinant technology as a fusion to a target protein. The smaller C-intein (Ic) carrying a quencher and a fluorophore, Q-Ic-Fl, was chemically synthesized and delivered into mammalian cells. We first tested fluorescent labeling of model membrane anchored proteins on live cells using engineered Npu DnaE split-intein based PTS reaction. The labeling reaction occurred in two different modes. First, the fast and instant labeling occurred via specific binding of two split-intein fragments, and second, more stable and irreversible labeling occurred via covalent bond formation through split-intein mediated PTS reaction. We next showed fluorescent labeling of cytosolic protein using a fluorescence-quenched peptide, Q-Ic-Fl. This approach enabled wash-free labeling of target cytosolic proteins. Finally, we attempted to photo-trigger the labeling reaction in live cells using Q-iso Ic-CPP-Fl. The photo cage was removed by irradiation to restore functional intein from the photocaged iso-Ic. The activated intein carried out the labeling reaction, successfully. This approach can be used as a tool to monitor the functions and locations of target proteins in live cells with spatiotemporal resolution.
E-mail dmldysl@gmail.com