Development of an Economical And Highly Productive Cell-Free Protein Synthesis System from E.coli Extracts

Despite the promising potential of cell-free protein synthesis, the high cost of the required reagents has plagued its practical application. In particular, most of the reagent cost is attributed to the expenses for conventional energy sources such as phophsoenol pyruvate (PEP) and creatine phopshate (CP). In this study, for the preparative expression of recombinant proteins at a reduced energy cost, we attempted to use the glycolytic intermediates to drive the cell-free protein synthesis in a CECF (continuous-exchange cell-free protein synthesis) reaction.Among the glycolytic intermediates examined, use of fructose 1,6-bisphosphate (FBP) gave the highest yield of protein synthesis, producing approximately 7 mg/ml of a CAT (chloramphenicol acetyl transferase) protein. We also found that protein synthesis in the CECF reaction was able to be directed by PCR-amplified linear DNA templates when by employing the measures tostabilize mRNA in the reaction mixture. As a result, protein synthesis reaction continued over 10 hours, producing 5 mg/ml of CAT.