Evolution of translation initiation sequences using in vitro yeast ribosome display

Rui Gan, Michael C. Jewett*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

We report a novel in vitro yeast ribosome display method based on cell-free protein synthesis (CFPS) using linear DNA templates. We demonstrate that our platform can enrich a target gene from a model library by 100-fold per round of selection. We demonstrate the utility of our approach by evolving cap-independent translation initiation (CITI) sequences, which result in a 13-fold increase in CFPS yields after four rounds of selection, and a threefold further increase by placing the beneficial short sequences in tandem. We also show that 12 of the selected CITI sequences permit precise control of gene expression in vitro over a range of up to 80-fold by enhancing translation (and not as cryptic promoters). These 12 sequences are then shown to tune protein expression in vivo, though likely due to a different mechanism. Looking forward, yeast ribosome display holds promise for evolving libraries of proteins and DNA regulatory parts for protein engineering and synthetic biology. Biotechnol. Bioeng. 2016;113: 1777–1786.

Original languageEnglish (US)
Pages (from-to)1777-1786
Number of pages10
JournalBiotechnology and Bioengineering
Volume113
Issue number8
DOIs
StatePublished - Aug 1 2016

Keywords

  • cap-independent translation initiation
  • directed evolution
  • ribosome display
  • synthetic biology
  • yeast cell-free protein synthesis

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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