Cell-free synthesis of glycoproteins
Glycoproteins have become an important focus of the current efforts in structural and functional genomics. In contrast, the molecular analysis of posttranslationally modified proteins lags far behind that of non-modified, cytosolic and soluble proteins. Preparing high quality samples of functionally folded glycoproteins represents a major bottleneck that restricts further structural and functional studies.
Cell-free protein synthesis 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 posttranslationally modified proteins. 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 cell lysates. 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 produced proteins.