Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses IZI-BB
Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses IZI-BB

Eukaryotic Lysates

Eukaryotische Lysate
© Fraunhofer IZI-BB

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.

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  • 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

 

 

 

 

 

 

  • Knauer, J. F., Liers, C., Hahn, S., Wuestenhagen, D. A., Zemella, A., Kellner, H., Haueis, L., Hofrichter, M., & Kubick, S. (2022). Cell-free production of the bifunctional glycoside hydrolase GH78 from Xylaria polymorpha. Enzyme and Microbial Technology, 161, 110110. https://doi.org/10.1016/j.enzmictec.2022.110110
  • Krebs, S. K., Stech, M., Jorde, F., Rakotoarinoro, N., Ramm, F., Marinoff, S., Bahrke, S., Danielczyk, A., Wüstenhagen, D. A., & Kubick, S. (2022). Synthesis of an Anti-CD7 Recombinant Immunotoxin Based on PE24 in CHO and E. coli Cell-Free Systems. International Journal of Molecular Sciences, 23(22). https://doi.org/10.3390/ijms232213697
  • Dhandapani, P., Dondapati, S. K., Zemella, A., Bräuer, D., Wüstenhagen, D. A., Mergler, S., & Kubick, S. (2021). Targeted esterase-induced dye (TED) loading supports direct calcium imaging in eukaryotic cell-free systems. RSC Advances, 11(27), 16285–16296. https://doi.org/10.1039/d0ra08397f
  • Wüstenhagen, D. A., Lukas, P., Müller, C., Aubele, S. A., Hildebrandt, J.-P., & Kubick, S. (2020). Cell-free synthesis of the hirudin variant 1 of the blood-sucking leech Hirudo medicinalis. Scientific Reports, 10(1), 19818.  https://doi.org/10.1038/s41598-020-76715-w
  • Thoring, L., Wüstenhagen, D. A., Borowiak, M., Stech, M., Sonnabend, A., & Kubick, S. (2016). Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of "Difficult-to-Express" Proteins and Future Perspectives. PloS One, 11(9), e0163670. dx.doi.org/10.1371/journal.pone.0163670 Artikel
  • Dondapati, S. K., Kreir, M., Quast, R. B., Wüstenhagen, D. A., Brüggemann, A., Fertig, N., & Kubick, S. (2014). Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system. Biosensors & Bioelectronics, 59, 174–183. https://doi.org/10.1016/j.bios.2014.03.004
  • Stech, M., Quast, R. B., Sachse, R., Schulze, C., Wüstenhagen, D. A., & Kubick, S. (2014). A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells. PloS One, 9(5), e96635. https://doi.org/10.1371/journal.pone.0096635
  • Bechlars, S., Wüstenhagen, D. A., Drägert, K., Dieckmann, R., Strauch, E., & Kubick, S. (2013). Cell-free synthesis of functional thermostable direct hemolysins of Vibrio parahaemolyticus. Toxicon : Official Journal of the International Society on Toxinology, 76, 132–142. https://doi.org/10.1016/j.toxicon.2013.09.012
  • Brödel, A. K., Sonnabend, A., Roberts, L. O., Stech, M., Wüstenhagen, D. A., & Kubick, S. (2013). Ires-mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems. PloS One, 8(12), e82234. https://doi.org/10.1371/journal.pone.0082234
  • Sachse, R., Wüstenhagen, D., Šamalíková, M., Gerrits, M., Bier, F. F., & Kubick, S. (2013). Synthesis of membrane proteins in eukaryotic cell‐free systems. Engineering in Life Sciences, 13(1), 39–48. https://doi.org/10.1002/elsc.201100235.
  • Zampatis, D. E., Rutz, C., Furkert, J., Schmidt, A., Wüstenhagen, D., Kubick, S., Tsopanoglou, N. E., & Schülein, R. (2012). The protease-activated receptor 1 possesses a functional and cleavable signal peptide which is necessary for receptor expression. FEBS Letters, 586(16), 2351–2359. https://doi.org/10.1016/j.febslet.2012.05.042
  • Brödel, A. K., Sonnabend, A., & Kubick, S. (2014). Cell-free protein expression based on extracts from CHO cells. Biotechnology and Bioengineering, 111(1), 25–36. https://doi.org/10.1002/bit.25013
  • Stech, M., Merk, H., Schenk, J. A., Stöcklein, W. F. M., Wüstenhagen, D. A., Micheel, B., Duschl, C., Bier, F. F., & Kubick, S. (2012). Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system. Journal of Biotechnology, 164(2), 220–231. https://doi.org/10.1016/j.jbiotec.2012.08.020