Figure 1.

General principle of eukaryotic cell-free technology for research and therapeutical applications. For eukaryotic cell-free protein synthesis, a suitable DNA template is required, which can be directly prepared from cellular mRNA by RT-PCR. In this way, 5′ and 3′ regulatory sequences (T7 promotor, T7 terminator, stem loops and hairpin sequences) are added to the DNA template. Alternatively, plasmids harboring regulatory sequences can be used for eukaryotic cell-free protein synthesis. The DNA template is transcribed into mRNA using T7 RNA polymerase (T7 Pol) directly added to the cell-free synthesis reaction. Eukaryotic cell-free protein synthesis is based on a eukaryotic cell lysate including endogenous microsomes derived from endoplasmic reticulum. Special eukaryotic lysates like wheat germ and rabbit reticulocyte lysate do not include endogenous microsomes. The eukaryotic cell lysate is supplemented with previously produced mRNA and buffer and energy components to perform cell-free protein synthesis. Applications of eukaryotic cell-free protein synthesis are the development of novel screening platforms for drugs, the functional characterization of proteins and the production of biotherapeutics.