Eukaryotic Lysates

Translational lysates form the foundation for the synthesis of proteins in cell-free systems. In order to ensure a correct translocation to the microsome membrane, endogenous microsomes in eukaryotic lysates are essential for the mapping of posttranslationally modified proteins, such as glycoproteins and membrane-bound proteins. The Eukaryotic Lysate Working Group has many years of experience in the cultivation of eukaryotic cell lines and their conversion to translational lysates for protein synthesis. Testing new cell lines for their capacity for in-vitro expression plays a large role. The development and continuous optimization of eukaryotic cell-free translation systems is an important research focus of the Working Group. The effect of fermentation and cell disruption as well as the transcription and translation components for the productivity of lysates are of key interest in this regard.

Validation of Protein Synthesis

During an evaluation of protein syntheses in cell-free systems, the templates’ overall capacity for expression is examined. A comparative screening in different systems (lysates based on E. coli cells, cultivated insects, CHO and human cell lines, as well as wheat germ lysates) can be carried out for this purpose in order to identify the ideal system for synthesizing the target protein. In addition to plasmids, linear constructs, such as PCR products or even direct mRNA, can be used as templates. Various reaction formats are available for the protein synthesis in order to maximize protein yield. The protein syntheses can be standardized and carried out according to GLP guidelines.

Fermentation of Eukaryotic Cell Lines

Eukaryotic cell lysates form the basis for cell-free protein synthesis. To obtain these in translationally active form, specific cell lines are cultivated under appropriate conditions. Consistent quality of the lysates for the cell-free synthesis of proteins is achieved by ensuring ideal conditions for growing the cell lines: as a suspension culture in chemically defined media in fermenters and using standardized production protocols. For the fermentation of the cell lines, various reaction procedures can be carried out (batch, repeated batch or continuous reaction processes (perfusion)), modified specifically for the cell line.

  • Fermentation of eukaryotic cell lines in a suspension culture
  • Production of translationally active lysates and their integration into cell-free systems
  • Development of eukaryotic cell-free translation systems
  • Testing of new cell lines for their in-vitro expression capacity
  • Validation of DNA and mRNA templates
  • Integration of regulatory sequences, signal peptides, IRES sites, purification and fluorescence tags through the generation of linear templates
  • Cell-free synthesis of hard-to-express proteins, such as cytotoxic proteins and membrane proteins
  • Evaluation of protein synthesis using various cell-free systems (lysates from insect cells, CHO cells, cultivated human cells, E. coli and wheat germ lysates) in batch and dialysis mode (CECF)
  • MS analyses of peptides, proteins (identification, characterization and testing of modifications, such as glycosylation, phosphorylation, palmitylation), protein-protein as well as protein-ligand interactions
  • Measuring synthesis yield using (14C) protein labeling and TCA precipitation
  • Characterization of protein synthesis with gel electrophoresis, autoradiography and quantitative imaging in phosphorimager protein analysis using fluorescence microscope and western blotting

  • Thoring L, Wüstenhagen DA, Borowiak M, Stech M, Sonnabend A, Kubick S. Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of »Difficult-to-Express« Proteins and Future Perspectives. PLoS One. 2016 Sep 29;11(9):e0163670. DOI article
  • Dondapati SK, Kreir M, Quast RB, Wüstenhagen DA, Brüggemann A, Fertig N, Kubick S. Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system. Biosensors & bioelectronics 59C(2014) 174-183.
  • Stech M, Quast RB, Sachse R, Schulze C, Wüstenhagen DA, Kubick S. A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells. PLoS One 9 (2014) e96635.
  • Bechlars S, Wüstenhagen DA, Drägert K, Dieckmann R, Strauch E, Kubick S. Cell-free synthesis of functional thermostable direct hemolysins of Vibrio parahaemolyticus. Toxicon 76 (2013) 132-142.
  • Brödel AK, Sonnabend A, Roberts L, Stech M, Wüstenhagen DA, Kubick S. IRES-Mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems. PLoS One 8 (2013) e82234.
  • Sachse R, Wüstenhagen D, Samalikova M, Gerrits M, Bier FF, Kubick S. Synthesis of membrane proteins in eukaryotic cell-free systems. Engineering in Life Sciences 13 (2013) 39-48.
  • Zampatis DE, Rutz R, Furkert J, Schmidt A, Wüstenhagen D, Kubick S, Tsopanoglou NE, Schülein R. The protease-activated receptor 1 possesses a functional and cleavable signal peptide which is necessary for receptor expression. FEBS letters 586(16) (2012); :2351-9
  • Brödel AK, Sonnabend A, Kubick S. Cell-free protein expression based on extracts from CHO cells. Biotechnology and Bioengineering 111 (2013) 25-36.
  • Stech M, Merk H, Schenk JA, Stöcklein W, Wüstenhagen DA, Micheel B, Duschl C, Bier FF, Kubick S. Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system. J Biotechnol. 164 (2012) 220-231.