Eukaryotic Lysates

Translation-active lysates form the basis for the synthesis of proteins in cell-free systems. Endogenous microsomes in eukaryotic lysates are essential to guarantee a correct translocation in the microsomal membrane to display post-translational modified proteins such as glycoproteins and membrane-based proteins. The Eukaryotic Lysates Unit has many years of experience in the cultivation of eukaryotic cell lines and their conversion into translation-active lysate for the synthesis of proteins. The evaluation of a new cell line’s capacity for in-vitro expression is of great significance. The development and continuous optimisation of eukaryotic cell-free translation systems is one of the unit's important research areas. The influence on lysate productivity of fermentation, cell disruption, and transcription and translation components is of crucial interest.

  • Fermentation of eukaryotic cell lines in suspension culture
  • Manufacture of translation-active lysates and their integration in cell-free systems
  • Development of eukaryotic cell-free translation systems
  • Test of new cell lines for their in-vitro expression ability
  • Validation of DNA and mRNA templates
  • Integration of regulatory sequences, signal peptides, IRES sites, cleaning procedures and fluorescence tags by generating linear templates
  • Cell-free synthesis of proteins that are difficult to express, such as cytotoxic proteins and membrane proteins
  • Evaluation of the protein synthesis when 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, characterisation and examination of modifications, such as glycosylations, phosphorylation, palmitylation), protein-protein as well as protein-ligand interactions
  • Determination of synthesis yield by means of (14C) protein labeling and TCA precipitation
  • Characterization of protein synthesis by gel electrophoresis, autoradiography and quantitative imaging in phosphorus imager protein analysis by means of fluorescence microscopy and Western blot

Fermentation of Eukaryotic Cell Lines

Eukaryotic cell lysates are the basis for cell-free protein synthesis. To obtain these cell lysates in translation-active form, defined cell lines are cultivated under suitable conditions. The optimal growth conditions of cell lines in a suspension culture in chemically-defined fermenter media using standardised manufacturing protocols ensures a uniform quality of the lysate for the cell-free synthesis of proteins. All relevant processes are deliberately scaled to liter volumes to also allow for cell-free synthesis of target-proteins in greater volumes. A continuous fermentation reaction, also called perfusion, was established for this purpose. As a consequence of the subsequent continuous reconditioning of the cells, large lysate batches of uniform quality can be obtained.

Development of New Eukaryotic Cell-Free Expression Systems

This project focuses on the continuous further development of existing systems as well as testing new cell lines for their general expression ability. The establishment of lysates based on mammalian cell lines is of particular significance for industrial protein production processes. Lysate from fermented CHO cells are currently being brought to market maturity.

Evaluation of Protein Syntheses in Cell-Free Systems

Every protein has its own requirements for optimal synthesis and protein folding that depend strongly on protein type. While the systems based on E. Coli or wheat germ lysates are well suited for cytosolic proteins, the synthesis of membrane proteins is more demanding. These proteins require membrane structures with an active translocon for an aligned integration into the biological membrane. The microsomes essential for this, based on vesicles of the endoplasmic reticulum, are contained in the lysates, based on eukaryotic cell lines due to the gentle breaking up of the cells. However, initiation and translation factors, chaperone and tRNA limitations also play important roles in the correct synthesis and protein folding.

An evaluation of protein synthesis in cell-free systems includes examination of the general expression ability of the templates. The examination can include a comparative screening in various systems (lysate based on E. Coli cells, cultivation insect/CHO/human cell lines as well as wheat germ lysates) to identify the optimal system for the synthesis of the target protein. Apart from plasmids as templates, linear constructs can also be used, such as PCR products containing regulatory elements such as T7 promoters, ribosome binding site etc., or mRNA can be directly employed. The reaction conditions in a defined system can be optimised to maximize protein yield.

Synthesis of Toxins

The synthesis of proteins using a cell-free method offers an innovative platform for relatively inexpensive, rapid and time-efficient analysis and pharmacological examinations of proteinogenic toxins. This can be done only with great effort and expense (if at all) on a cellular basis, since the synthesized protein has a toxic effect on the host cell, which then dies off. It can be shown that thermo-stable hemolysin (TDH), which occurs naturally in mussels and is responsible for a multitude of human intolerances, has cytotoxic activity if manufactured on a cell-free basis. The cloning-free production method based on PCR products permits a much more time-efficient synthesis of the toxin without the need for genetically modified organisms.

Cell Culture Laboratories of Safety Class S1

  • 5 l fermenter (Sartorius Biostat B DCU-II Advanced Additive Flow System; 2x 5 l chamber, upgradeable with up to 6 vessels between 1 and 10 l)
  • 30 l fermenter (Sartorius Biostat D DCU)
  • Confocal laser scanning microscope (Zeiss CLSM 510)
  • Automated cell assay and screening unit (PerkinElmer; CellLuxCellularFluorescence Workstation)

Isotope Laboratory

  • Protein labeling (interaction with 14C, 32P, 35S)
  • Exhaust systems for the separation of 14C labeled protein precipitates (TCA precipitation)
  • Scintillation counters (6500 Multi)
  • Gel drying unit for autoradiographs (Unigeldryer 3545)
  • Typhoon Trio+ variable-mode imager (radioactivity, fluorescence and chemiluminescence with extended 10 µm pixel scan)

Laboratories of the Safety Class S1 for Molecular-Biological Work

  • Multimode reader Berthold LB 941 vi-S TriStar (flash, glow and colour luminescence, absorption, fluorescence, FRET, BRET)
  • Sirius single tube illuminometer (Titertek Berthold)
  • Spectrophotometer for UV/Vis (Nanodrop ND-2000c)
  • Bioreactors for cell-free protein synthesis in batch process and dialysis scale

Mass Spectrometry in Laboratories of Safety Class S1

  • Mass spectrometer Q-TOF MaxIs Impact (Bruker Daltonics) with interchangeable ion sources (offline nanoESI source, conventional ESI source, captive spray for nano-LC coupling)
  • Ultra-sensitive ion trap AmaZon Speed ETD (Bruker Daltonics) with interchangeable ion sources (conventional ESI source, captive spray for the nano-LC coupling)
  • UHPLC chromatography systems, 3000 system (Dionex)

Analytics of Membrane Proteins in Laboratories of Safety Class S1

  • Patch-Clamp system "Port-A-Patc" and Orbit 16 from Nanion
  • Particle analysis by means of “Zetasizer Nano ZS” from Malvern

  • 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 dx.doi.org/10.1371/journal.pone.0163670 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.