A combined cell-free transcription-translation system from Saccharomyces cerevisiae for rapid and robust protein synthesis

Rui Gan, Michael C. Jewett*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

48 Scopus citations

Abstract

Cell-free protein synthesis (CFPS) provides a valuable platform for understanding, using, and expanding the capabilities of the translation apparatus. For example, high-throughput CFPS is helping to address the increasing discrepancy between genome sequence data and their translation products. Here, we report the development of a combined cell-free transcription-translation (Tx/Tl) system from Saccharomyces cerevisiae that is suitable for such efforts. First, we show the ability to enable translation initiation in a cap-independent manner. The performance of various genetic elements was assessed, including 5'-UTR, 3'-UTR, and length of poly(A) tail. A specific vector harboring the 5'-UTR fragment of the Ω sequence from the tobacco mosaic virus and a poly(A) tail of 50 nucleotides led to optimal performance. Second, we developed a simple, two-step polymerase chain reaction (PCR) method for high-throughput production of linear templates for yeast CFPS. This procedure allows all functional elements needed for Tx/Tl to be added to an open-reading frame directly by overlap extension PCR. Our two-step PCR method was successfully applied to three reporter proteins: luciferase, green fluorescence protein, and chloramphenicol acetyl transferase, yielding 7 to 12.5 μg mL-1 active protein after 1.5-h batch reactions. Surprisingly, the linear templates outperformed plasmid DNA by up to 60%. Hence, the presented CFPS method has the potential to rapidly prepare tens to thousands of DNA templates without time-consuming cloning work. Further, it holds promise for fast and convenient optimization of expression constructs, study of internal ribosome entry site, and production of protein libraries for genome-scale studies.

Original languageEnglish (US)
Pages (from-to)641-651
Number of pages11
JournalBiotechnology Journal
Volume9
Issue number5
DOIs
StatePublished - May 2014

Keywords

  • Cell-free protein synthesis
  • Combined transcription-translation
  • In vitro
  • Yeast

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Molecular Medicine

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