| Characterization of Cell Protein Translocation using Enzyme Fragment Complementation |
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Presentation: 5C03 Session: Advanced Technology for Drug Discovery - Part 2Peter Fung, Kun Peng, Betty Bosano, Phil Kobel, Ganapathirama Raghu, Joe Horecka, Keith Olsen and Richard Eglen, DiscoveRx Corp. Presenting Author: Richard Eglen, DiscoveRx Corp - USA β Galactosidase (β gal) can be genetically divided into two fragments: - Enzyme Acceptor (EA), and a smaller fragment, Enzyme Donor (ED). These two fragments recombine to form competent β gal, via enzyme fragment complementation (EFC). Both EA and ED-fusion proteins can be expressed in mammalian cells, with expression of the former localized to either membrane or nucleus. 'Positional' complementation only occurs, therefore, when the ED: protein fusion translocates, providing homogeneous assays for interrogation of cell pathways resulting in translocation. Trafficking to the cell exterior can also be monitored using fusion proteins expressed in a suitable extracellular loop, flanked in series by consensus protease cleavage sites. Appearance on the cell surface is monitored by addition of a protease and subsequently detected by EFC. Since none of these methods, collectively, employ imaging, they are amenable to high throughput automation systems, enabling researchers to measure intracellular trafficking pathways using luminescence plate readers. |