Membrane Protein Synthesis in Giant Vesicles

Abstract

Interest in the development of biomimetic cell models is driven by its potential to go beyond a purely descriptive picture of cellular processes towards a quantitative understanding and rigorous validation of theoretical modeling. Cells are exceptionally complex entities with a large variety of active and passive components. A quantitative investigation of a specific process in such complex environment is prohibitively challenging. In a biomimetic system like a giant unilamellar vesicle on the other hand the key components of a specific process can be studied isolated from other factors. In this way, it is possible to achieve quantitative information and that will allow us to discover the governing principles of the processes involved.Recently we introduced a protocol to prepare advanced cell models from giant unilamellar vesicles for studies of membrane processes that involve transmembrane proteins. We further showed that specific functionalization permits to use those biomimetic systems in a lab-on-a-chip scenario. [1] Building on this development, we present here a novel approach that allows us to realize a mimetic cell model that includes in situ protein synthesis and active membrane translocation. Giant unilamellar vesicles were prepared starting from eukaryotic cell lysates containing both the eukaryotic protein synthesis machinery as well as the translocon that is required to integrate proteins into membranes. Our soft methodology for vesicle preparation on agarose-coated surfaces allowed us to keep the translocon fully functional. The advantage of in situ expression and translocation as described here to earlier attempts to produce giant proteo-liposomes is that all proteins are built into the vesicle membrane in the correct orientation. Our novel cell model opens up the possibility to study e.g. protein synthesis in vitro with single-molecule microscopy.[1] S. Fenz, R. Sachse, S. Kubick and T. Schmidt; BPJ 2012, 102(3), pp18a