Microsomes from Cell-free Systems as Functional Modules for GUV Formation Processes

View all posters

Rita Sachse, Susanne Fenz, Doreen Wüstenhagen, Thomas Schmidt and Stefan Kubick

Fraunhofer IBMT, Germany

The formation of well defined synthetic membrane model systems is commonly used in the field of biophysics to study basic biological processes in a less complex environment than a viable cell. However, the functionality of membrane proteins not only depends on their incorporation into a lipid bilayer, but also e.g. on subsequent posttranslational modification. The integration of these biological processes into synthetic systems is rather challenging. However, due to the fact that membrane proteins are naturally low abundant in living cells, common over-expression systems often reach their limits caused by cytotoxic effects or aggregation of membrane proteins. Thus, the reduction of the biological protein production machinery to a viable cell independent system is favourable. Our eukaryotic cell-free system based on insect cell lysates provides endoplasmic reticulum derived vesicles, so called microsomes, which enable co-translational translocation as well as posttranslational modifications as glycosylation or lipid modification. These microsomes, serving as micro-containers, enable us to integrate the membrane protein of interest into giant unilamellar vesicles (GUVs) using the electroswelling process [1]. Additionally, with this method we are able to simultaneously modify microsomes containing membrane proteins with synthetic lipids to gain so called hybrid-GUVs. Thus, techniques for functionalization and immobilization are applicable to embed membrane systems into technical processes or incorporate proteins in synthetically modified biological membranes. Ongoing research is focusing on mild formation methods, such as agarose-supported swelling to gain translocationally active GUVs for direct expression of active membrane proteins. Taken together, these methods now enable the engineering of biomimetic vesicles and the development of novel functional assays based on cell-free systems. [1] “Towards an artificial cell: Protein incorporation in giant unilamellar vesicles under physiological conditions”, Paige M. Shaklee, Stefan Semrau, Maurits Malkus, Stefan Kubick, Marileen Dogterom,Thomas Schmidt, ChemBioChem (2010)