Robust in vitro affinity maturation strategy based on interface-focused high-throughput mutational scanning

Yasuhiro Fujino, Risako Fujita, Kouichi Wada, Kotomi Fujishige, Takashi Kanamori, Lindsey Hunt, Yoshihiro Shimizu, Takuya Ueda

研究成果: Article査読

21 被引用数 (Scopus)

抄録

Development of protein therapeutics or biosensors often requires in vitro affinity maturation. Here we report a robust affinity engineering strategy using a custom designed library. The strategy consists of two steps beginning with identification of beneficial single amino acid substitutions then combination. A high quality combinatorial library specifically customized to a given binding-interface can be rapidly designed by high-throughput mutational scanning of single substitution libraries. When applied to the optimization of a model antibody Fab fragment, the strategy created a diverse panel of high affinity variants. The most potent variant achieved 2110-fold affinity improvement to an equilibrium dissociation constant (Kd) of 3.45. pM with only 7 amino acid substitutions. The method should facilitate affinity engineering of a wide variety of protein-protein interactions due to its context-dependent library design strategy.

本文言語English
ページ(範囲)395-400
ページ数6
ジャーナルBiochemical and Biophysical Research Communications
428
3
DOI
出版ステータスPublished - 2012 11 23
外部発表はい

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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