Purification and Functional Reconstitution of the Bacterial Protein Translocation Pore, the SecYEG Complex
In bacteria, proteins are secreted across the cytoplasmic membrane by a protein complex termed translocase. The ability to study the activity of the translocase in vitro using purified proteins has been instrumental for our understanding of the mechanisms underlying this process. Here, we describe the protocols for the purification and reconstitution of the SecYEG complex in an active state into liposomes. In addition, fluorescence based in vitro assays are described that allow monitoring translocation activity discontinuously and in real time.
Key wordsProtein secretion reconstitution SecYEG SecA in vitro translocation fluorescence quenching
This work was supported by NanoNed, a national nanotechno-logy program coordinated by the Dutch Ministry of Economical Affairs and the Zernike Institute for Advanced Materials.
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