We modified a cell‐free coupled transcription/translation system from Escherichia coli with the T7 phage RNA polymerase, and achieved a productivity as high as 0.4 mg protein/ml reaction mixture. First, we found that the optimal concentrations of phosphoenolpyruvate and poly(ethylene glycol) are interdependent; higher concentrations of the former should be used at higher concentrations of the latter. Second, the use of a condensed 30000×g cell extract, in place of the conventional one, significantly increased the initial rate of protein synthesis. This phenomenon was demonstrated to be due to a reason other than elimination of inhibitory molecule(s) from the extract. For this system with the condensed extract, the phosphoenolpyruvate and poly(ethylene glycol) concentrations were again co‐optimized, resulting in production of chloramphenicol acetyltransferase at a productivity of 0.3 mg/ml. Finally, the productivity was further increased up to 0.4 mg/ml, by supplementation of the pool of amino acids. This improved cell‐free protein synthesis system is superior in productivity to any other cell‐free systems.

Factors causing the early cessation of protein synthesis have been studied in a cell‐free system from Escherichia coli. We discovered that phosphoenol pyruvate (PEP), the secondary energy source for ...

This report describes a novel and simple cell‐free protein synthesis system. In this paper, we prove that the short duration of protein synthesis in a conventional cell‐free protein synthesis system ...