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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

26 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)395-400
Number of pages6
JournalBiochemical and Biophysical Research Communications
Issue number3
Publication statusPublished - 2012 Nov 23
Externally publishedYes


  • Affinity maturation
  • Antibody Fab fragment
  • High-throughput sequencing
  • Mutation scanning
  • Protein engineering
  • Ribosome display

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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