A HIGHLY EFFICIENT CELL-FREE PROTEIN-SYNTHESIS SYSTEM FROM ESCHERICHIA-COLI

We modified a cell-free coupled transcription/translation system from Escherichia coli with the T7 phage RNA polymerase, and achieved a prod uctivity as high as 0.4 mg protein/ml reaction mixture. First, we foun d that the optimal concentrations of phosphoenolpyruvate and poly(ethy lene glycol) are interdependent; higher concentrations of the former s hould be used at higher concentrations of the latter. Second, the use of a condensed 30 000Xg 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 eliminati on of inhibitory molecule(s) from the extract. For this system with th e condensed extract, the phosphoenolpyruvate and poly(ethylene glycol) concentrations were again co-optimized, resulting in production of ch loramphenicol acetyltransferase at a productivity of 0.3 mg/ml. Finall y, the productivity was further increased up to 0.4 mg/ml, by suppleme ntation of the pool of amino acids. This improved cell-free protein sy nthesis system is superior in productivity to any other cell-free syst ems reported so far, including the continuous-flow cell-free system.