Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis

Biotechnol Bioeng. 2004 Apr 5;86(1):19-26. doi: 10.1002/bit.20026.


Cell-free translation systems generally utilize high-energy phosphate compounds to regenerate the adenosine triphosphate (ATP) necessary to drive protein synthesis. This hampers the widespread use and practical implementation of this technology in a batch format due to expensive reagent costs; the accumulation of inhibitory byproducts, such as phosphate; and pH change. To address these problems, a cell-free protein synthesis system has been engineered that is capable of using pyruvate as an energy source to produce high yields of protein. The "Cytomim" system, synthesizes chloramphenicol acetyltransferase (CAT) for up to 6 h in a batch reaction to yield 700 microg/mL of protein. By more closely replicating the physiological conditions of the cytoplasm of Escherichia coli, the Cytomim system provides a stable energy supply for protein expression without phosphate accumulation, pH change, exogenous enzyme addition, or the need for expensive high-energy phosphate compounds.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, U.S. Gov't, P.H.S.
  • Validation Study

MeSH terms

  • Adenosine Triphosphate / chemistry
  • Adenosine Triphosphate / metabolism
  • Biomimetics / methods*
  • Cell-Free System / chemistry
  • Cell-Free System / metabolism
  • Chloramphenicol O-Acetyltransferase / biosynthesis*
  • Chloramphenicol O-Acetyltransferase / chemical synthesis
  • Chloramphenicol O-Acetyltransferase / genetics
  • Cytoplasm / chemistry
  • Cytoplasm / metabolism*
  • Enzyme Activation
  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Homeostasis / physiology
  • Hydrogen-Ion Concentration
  • Protein Engineering / methods*
  • Pyruvates / metabolism
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / chemistry


  • Pyruvates
  • Recombinant Proteins
  • Adenosine Triphosphate
  • Chloramphenicol O-Acetyltransferase