Substrate replenishment extends protein synthesis with an in vitro translation system designed to mimic the cytoplasm

Michael C. Jewett, James R. Swartz*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

86 Scopus citations


Cytoplasmic mimicry has recently led to the development of a novel method for cell-free protein synthesis called the "Cytomim" system. In vitro translation with this new system produced more than a 5-fold yield increase of chloramphenicol acetyl transferase (CAT) relative to a conventional method using pyruvate as an energy substrate. Factors responsible for activating enhanced protein yields, and causes leading to protein synthesis termination have been assessed in this new system. Enhanced yields were caused by the combination of three changes: growing the extract source cells on 2x YTPG media versus 2x YT, replacing polyethylene glycol with spermidine and putrescine, and reducing the magnesium concentration from conventional levels. Cessation of protein synthesis was primarily caused by depletion of cysteine, serine, CTP, and DTP. Substrate replenishment of consumed amino acids, CTP, and DTP extended the duration of protein synthesis to 24 h in fed-batch operation and produced 1.2 mg/mL of CAT. By also adding more T7 RNA polymerase and plasmid DNA, yields were further improved to 1.4 mg/mL of CAT. These results underscore the critical role that nucleotides play in the combined transcription-translation reaction and highlight the importance of understanding metabolic processes influencing substrate depletion.

Original languageEnglish (US)
Pages (from-to)465-471
Number of pages7
JournalBiotechnology and Bioengineering
Issue number4
StatePublished - Aug 20 2004


  • Amino acid
  • Cell-free protein synthesis
  • Combined transcription-translation
  • Cytoplasmic mimicry
  • Fed-batch reaction
  • Nucleotide

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology


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