A highly efficient cell-free protein synthesis system from Escherichia coli

Eur J Biochem. 1996 Aug 1;239(3):881-6. doi: 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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell-Free System* / drug effects
  • Chloramphenicol O-Acetyltransferase / biosynthesis*
  • DNA-Directed RNA Polymerases / metabolism
  • Escherichia coli / metabolism*
  • Magnesium / pharmacology
  • Phosphoenolpyruvate / pharmacology
  • Polyethylene Glycols / pharmacology
  • Protein Biosynthesis*
  • Transcription, Genetic*
  • Viral Proteins

Substances

  • Viral Proteins
  • Polyethylene Glycols
  • Phosphoenolpyruvate
  • Chloramphenicol O-Acetyltransferase
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases
  • Magnesium