Substrate replenishment and byproduct removal improve yeast cell-free protein synthesis

Jennifer A. Schoborg, C. Eric Hodgman, Mark J. Anderson, Michael C. Jewett*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

38 Scopus citations


Cell-free protein synthesis (CFPS) platforms are now considered a powerful tool for synthesizing a variety of proteins at scales from pL to 100 L with accelerated process development pipelines. We previously reported the advancement of a novel yeast-based CFPS platform. Here, we studied factors that cause termination of yeast CFPS batch reactions. Specifically, we characterized the substrate and byproduct concentrations in batch, fed-batch, and semi-continuous reaction formats through high-performance liquid chromatography (HPLC) and chemical assays. We discovered that creatine phosphate, the secondary energy substrate, and nucleoside triphosphates were rapidly degraded during batch CFPS, causing a significant drop in the reaction's energy charge (E.C.) and eventual termination of protein synthesis. As a consequence of consuming creatine phosphate, inorganic phosphate accumulated as a toxic byproduct. Additionally, we measured amino acid concentrations and found that aspartic acid was rapidly consumed. By adopting a semi-continuous reaction format, where passive diffusion enables substrate replenishment and byproduct removal, we achieved over a 70% increase in active superfolder green fluorescent protein (sfGFP) as compared with the batch system. This study identifies targets for the future improvement of the batch yeast CFPS reaction. Moreover, it outlines a detailed, generalized method to characterize and improve other CFPS platforms.

Original languageEnglish (US)
Pages (from-to)630-640
Number of pages11
JournalBiotechnology Journal
Issue number5
StatePublished - May 2014


  • Cell-free biology
  • Cell-free protein synthesis
  • In vitro translation
  • Protein expression
  • Saccharomyces cerevisiae

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Molecular Medicine


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