A Highly Efficient Cell‐Free Protein Synthesis System from Escherichia coli
Open Access
- 1 August 1996
- journal article
- Published by Wiley in European Journal of Biochemistry
- Vol. 239 (3), 881-886
- https://doi.org/10.1111/j.1432-1033.1996.0881u.x
Abstract
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.Keywords
This publication has 38 references indexed in Scilit:
- Lounging in a lysosome: the intracellular lifestyle of Coxiella burnetiiCellular Microbiology, 2007
- Valyl‐tRNA Synthetase from ArtemiaEuropean Journal of Biochemistry, 1995
- In vitro protein engineering using synthetic tRNAAla with different anticodonsBiochemistry, 1993
- Probing the Structure and Mechanism of Ras Protein with an Expanded Genetic CodeScience, 1993
- De novo design, synthesis and study of albebetin, a polypeptide with a predetermined three-dimensional structure: Probing the structure at the nanogram levelJournal of Molecular Biology, 1992
- A General Method for Site-specific Incorporation of Unnatural Amino Acids into ProteinsScience, 1989
- Effect of polypeptide initiation factors on the spermidine stimulation of initiation complex formationBiochemical and Biophysical Research Communications, 1980
- Cell-free translation of messenger RNA coding for a precursor of human calcitoninBiochemical and Biophysical Research Communications, 1979
- The Interdependence of Magnesium with Spermidine and Phosphoenolpyruvate in an Enzyme‐Synthesizing System in vitroEuropean Journal of Biochemistry, 1976
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970