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 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 x 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 reported so far, including the continuous-flow cell-free system.
Kim
D M
DM
Interdisciplinary Program for Biochemical Engineering and Technology, College of Engineering, Seoul National University, Korea.
Kigawa
T
T
Choi
C Y
CY
Yokoyama
S
S
D002474:Cell-Free System / Q000187:drug effects; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D014176:Protein Biosynthesis; D014158:Transcription, Genetic; D014764:Viral Proteins
D014764:Viral Proteins; D011092:Polyethylene Glycols; D010728:Phosphoenolpyruvate; D015500:Chloramphenicol O-Acetyltransferase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium