Efficient protein selection based on ribosome display system with purified components

Hiroyuki Ohashi, Yoshihiro Shimizu, Bei Wen Ying, Takuya Ueda

Research output: Contribution to journalArticle

98 Citations (Scopus)

Abstract

Using the PURE (Protein synthesis Using Recombinant Elements) system, we developed an efficient and highly controllable ribosome display method for selection of functional protein. The PURE system is composed of purified factors and enzymes that are responsible for gene expression in Escherichia coli. We performed the detailed analyses and optimization of the ribosome display system and demonstrated the formation of stable mRNA/ribosome/polypeptide ternary complexes. As complex formation is fundamental to successful ribosome display, these improvements resulted in a dramatic increase in the mRNA recovery rate. As a result, a ∼12,000-fold enrichment of single-chain antibody (scFv) cDNA was achieved in a single round of selection. Specific selection of scFv mRNA from a 1:1010 dilution in competitor mRNA was achieved with only three rounds of affinity selection. These findings, together with the results in the accompanying paper [T. Matsuura, H. Yanagida, J. Ushioda, I. Urabe, T. Yomo, Nascent chain, RNA, and ribosome complexes generated by pure translation system (see the accompanying paper).], demonstrate that the PURE system can provide a basis for reliable and reproducible ribosome display.

Original languageEnglish
Pages (from-to)270-276
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume352
Issue number1
DOIs
Publication statusPublished - 2007 Jan 5
Externally publishedYes

Keywords

  • Antibody
  • Cell-free protein synthesis system
  • In vitro selection
  • In vitro translation
  • Molecular evolution
  • PURE system
  • Protein engineering
  • Ribosome display
  • Translation
  • scFv

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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