TY - JOUR
T1 - Efficient protein selection based on ribosome display system with purified components
AU - Ohashi, Hiroyuki
AU - Shimizu, Yoshihiro
AU - Ying, Bei Wen
AU - Ueda, Takuya
N1 - Funding Information:
We thank Dr. Hiroshi Ueda (The University of Tokyo) for providing the plasmid encoding scFv-HyHEL10. This work was supported in part by a Grants-in-Aid for Scientific Research (A) No. 14208080 (to T.U.) from the Ministry of Education, Science, Sports and Culture of Japan.
PY - 2007/1/5
Y1 - 2007/1/5
N2 - 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.
AB - 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.
KW - Antibody
KW - Cell-free protein synthesis system
KW - In vitro selection
KW - In vitro translation
KW - Molecular evolution
KW - PURE system
KW - Protein engineering
KW - Ribosome display
KW - Translation
KW - scFv
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U2 - 10.1016/j.bbrc.2006.11.017
DO - 10.1016/j.bbrc.2006.11.017
M3 - Article
C2 - 17113037
AN - SCOPUS:33751410139
VL - 352
SP - 270
EP - 276
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 1
ER -