Mimicking the Escherichia coli Cytoplasmic Environment Activates Long-Lived and Efficient Cell-Free Protein Synthesis

Michael C. Jewett, James R. Swartz*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

252 Scopus citations

Abstract

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 acetyl-transferase (CAT) for up to 6 h in a batch reaction to yield 700 μg/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.

Original languageEnglish (US)
Pages (from-to)19-26
Number of pages8
JournalBiotechnology and Bioengineering
Volume86
Issue number1
DOIs
StatePublished - Apr 5 2004

Keywords

  • Cell-free protein synthesis
  • Combined transcription-translation
  • Cytoplasmic mimicry
  • Phosphate and pH homeostasis
  • Polyamines
  • Pyruvate

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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