Cell-free Protein Synthesis

© Fraunhofer IME | Holger Spiegel

Cell-free protein synthesis (CFPS) means the in vitro production of proteins without the use of living cells. Instead, we use special cell extracts or lysates containing the protein synthesis apparatus and all further necessary components. In principle, such extracts can be prepared from any kind of cell. However, the most widely used cell-free systems are prepared from the bacterium Escherichia coli, wheat germ, rabbit reticulocytes or insect cells. Following the addition of amino acids, an energy source, salts and other cofactors, almost any protein can be synthesized from a DNA or RNA template.

Compared to cell-based expression, CFPS offers advantages such as shorter process times and the direct control and monitoring of reaction conditions. PCR products can be used directly for the simultaneous expression of multiple proteins without laborious cloning and transformation steps. CFPS platforms allow the addition of accessory factors that promote protein folding or the incorporation of unnatural amino acids. They also facilitate the expression of cytotoxic proteins that cannot be produced in living cells.

During a project funded by the Federal Ministry of Education and Research (BMBF) we developed a system based on tobacco BY-2 cell lysate (BYL) that is superior to commercially available systems in terms of target protein yield and flexibility.

Selected Publications

Buntru, M., Vogel, S., Spiegel, H., Schillberg, S., 2014. Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system. BMC Biotechnology 14, 37. http://doi.org/10.1186/1472-6750-14-37

Buntru, M., Vogel, S., Stoff, K., Spiegel, H., Schillberg, S., 2015. A versatile coupled cell-free transcription–translation system based on tobacco BY-2 cell lysates. Biotechnol. Bioeng. 112, 867–878. http://doi.org/10.1002/bit.25502