Enhancing the efficiency of cell-free protein synthesis system by systematic titration of transcription and translation components

Cell-free protein synthesis (CFPS) is a valuable tool for basic and applied research. The Escherichia coli cell lysate-based system is the most widely used for in vitro transcription and translation (TX-TL). To optimize this system and increase target protein production, the present study investigated the contribution of most of the macromolecular components of the TX-TL machinery by means of systematic titration experiments. A 1.9-fold increase in enhanced green fluorescent protein expression was achieved relative to the reference system by single-component supplementation, while a 2.5-fold increase was achieved by adding T7 RNA polymerase, ribosome-recycling factor, translation initial factor (IF) 1, IF2, and elongation factor Tu. A similar enhancement in the production of other proteins was also achieved by supplementation, thereby indicating the broad applicability of this approach. These results provide useful information for further optimization of CFPS and a basis for improving industrial enzyme production by engineering bacterial strains that express specific components of the TX-TL machinery.