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.

Moreover, we have expanded our cell-free protein synthesis system by the insertion of orthogonal tRNA/synthetase pairs to facilitate the cotranslational and site directed incorporation of non-canonical building blocks. These fluorescently labeled and chemoselective moieties enable the site-specific modification of de novo synthesized glycoproteins.

Dr. Stefan Kubick
Head of Department
Cell-Free and Cell-Based Bioproduction
Fraunhofer Institute for Cell Therapy and Immunology (IZI)
Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam
Science Campus Golm
Am Mühlenberg 13
14476 Potsdam

Phone: +49 (0)331-58187.306


Dr. Stefan Kubick
Dr. Stefan Kubick
Dr. Stefan Kubick simultaneously heads the Department of „Cell-free Bioproduction“ at the Frauenhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses Potsdam-Golm (Fraunhofer IZI-BB) and leads the group „In vitro Protein Labeling“. His laboratory exploits cell-free protein synthesis as a versatile tool for functional genomics, e.g. cell-free synthesis of membrane proteins and glycoproteins, as well as chip-based protein synthesis and translational regulation. Dr. Kubick is also a lecturer at the Free University of Berlin and the University of Potsdam. He is an affiliate of the Technical University of Berlin and Lecturer at the University of Applied Sciences, Berlin, Germany. As a chairman of the newly founded network, glyconet Berlin Brandenburg, he actively works on establishing the region as a center of excellence in glycobiology.