Efficient protein selection based on ribosome display system with purified components

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

研究成果: Article査読

100 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)270-276
ページ数7
ジャーナルBiochemical and Biophysical Research Communications
352
1
DOI
出版ステータスPublished - 2007 1 5
外部発表はい

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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