Stefan Kubick: "Cell-free Synthesis of Posttranslationally Modified Membrane Proteins"

Stefan Kubick
Fraunhofer-Institut für Biomedizinische Technik, IBMT, Potsdam

Cell-free Synthesis of Posttranslationally Modified Membrane Proteins

Membrane proteins have become an important focus of the current efforts in structural and functional genomics and the rapid progress of various genome sequencing projects has greatly accelerated the discovery of novel genes encoding membrane proteins. In contrast, the molecular analysis of membrane proteins lags far behind that of cytosolic soluble proteins. Preparing high quality samples of functionally folded proteins represents a major bottleneck that restricts further structural and functional studies. Cell-free protein expression systems, in particular those of eukaryotic origin, have recently been developed as promising tools for the rapid and efficient production of a wide variety of membrane proteins. A large number of these proteins, however, require posttranslational modifications for optimum function. Several membrane proteins have been expressed in vivo to date, most of them being functionally, antigenically, and immunogenically similar to their authentic counterparts. This is mainly due to the properties of cultured eukaryotic cells, which are able to carry out many types of posttranslational modifications such as the addition of N- and O- linked oligosaccharides, but also palmitoylation, myristylation, and phosphorylation. Based on these versatile properties of cultured cell lines, we have developed a technique for the standardized production of translationally active eukaryotic lysates from insect cells. In contrast to other cell-free protein synthesis systems (e.g., rabbit reticulocyte lysates and wheat germ extracts) our homogenization procedure avoids any serious breakdown of membrane vesicles already existing in the cytoplasm of the prepared eukaryotic cells. We have demonstrated the functional integrity of these subcellular components by showing signal peptide cleavage as well as glycosylation of in vitro expressed membrane proteins.

The development of this novel eukaryotic in vitro translation system now expands the possibilities of cell-free protein synthesis, since posttranslational modifications significantly alter the physical and chemical properties of proteins, including their folding and conformational distribution and these modifications are frequently a fundamental prerequisite for functional activity.