Limiting factors of the translation machinery

Cell-free production of proteins is a rapid developing technology for many applications in proteomic sciences. Although great progress was made in the last years, in vitro protein synthesis is still less efficient than in vivo protein synthesis. For Escherichia coli based systems, all factors needed for cell-free protein synthesis are established, as shown in the reconstituted PURE system. Here we report the influence of additional translation factors, aminoacyl-tRNA synthetases and translational active ribosomes on cell-free protein synthesis of E. coli S30 extracts, indicating that none of these factors is a limiting factor for the protein synthesis. Furthermore, we show that varying the ratio of ribosomes including associated factors to other factors present in the extracts could not increase the yield of protein synthesized. In summary our results provide strong evidence for an optimal reconstitution of the translation machinery in E. coli S30 extracts.