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

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

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
Volume428
Issue number3
DOIs
Publication statusPublished - 2012 Nov 23
Externally publishedYes

Fingerprint

Libraries
Substitution reactions
Throughput
Amino Acid Substitution
Scanning
Amino Acids
Immunoglobulin Fragments
Immunoglobulin Fab Fragments
Proteins
Biosensing Techniques
Biosensors
Antibodies
In Vitro Techniques
Therapeutics

Keywords

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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Robust in vitro affinity maturation strategy based on interface-focused high-throughput mutational scanning. / Fujino, Yasuhiro; Fujita, Risako; Wada, Kouichi; Fujishige, Kotomi; Kanamori, Takashi; Hunt, Lindsey; Shimizu, Yoshihiro; Ueda, Takuya.

In: Biochemical and Biophysical Research Communications, Vol. 428, No. 3, 23.11.2012, p. 395-400.

Research output: Contribution to journalArticle

Fujino, Yasuhiro ; Fujita, Risako ; Wada, Kouichi ; Fujishige, Kotomi ; Kanamori, Takashi ; Hunt, Lindsey ; Shimizu, Yoshihiro ; Ueda, Takuya. / Robust in vitro affinity maturation strategy based on interface-focused high-throughput mutational scanning. In: Biochemical and Biophysical Research Communications. 2012 ; Vol. 428, No. 3. pp. 395-400.
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