Fermenter im Labor
© Fraunhofer IZI-BB
Fermenter im Labor

Fermentation of eukaryotic cells

Eukaryotic cell lysates are the basis for cell-free protein synthesis. To obtain translational-active lysates, defined cell lines are cultivated under appropriate conditions. Optimal growth conditions of the cell lines as suspension culture in chemically defined media in fermenters and standardized production protocols ensure a constant quality of the lysates for the cell-free synthesis of proteins. For the fermentation of the cell lines, different reaction runs (batch, repeated batch or continuous reaction runs (perfusion)), adapted to the cell line, can be performed.

Methods

  • Fermentation of eukaryotic cell lines in suspension culture
  • Production of translational lysates and their integration into cell-free systems
  • Development of eukaryotic cell-free translation systems
  • Testing new cell lines for their in vitro expression ability
  • Validation of DNA and mRNA templates
  • Integration of regulatory sequences, signal peptides, IRES sites, purification and fluorescence tags via linear template generation
  • Cell-free synthesis of proteins that are difficult to express, such as cytotoxic proteins and membrane proteins
  • Evaluation of protein synthesis using different cell-free systems (lysates from insect cells, CHO cells, cultured human cells; E. coli and wheat germ lysates) in batch and dialysis mode (CECF)
  • Determination of the synthesis yield using (14C)-protein labelling and TCA precipitation
  • Characterization of protein synthesis by gel electrophoresis, autoradiography and quantitative imaging in phosphorimager protein analysis by fluorescence microscopy and Western blotting