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

Biochem Biophys Res Commun. 2012 Nov 23;428(3):395-400. doi: 10.1016/j.bbrc.2012.10.066. Epub 2012 Oct 26.


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.

MeSH terms

  • Amino Acid Substitution
  • Combinatorial Chemistry Techniques*
  • Complementarity Determining Regions / genetics*
  • DNA Mutational Analysis / methods*
  • High-Throughput Nucleotide Sequencing / methods*
  • High-Throughput Screening Assays / methods*
  • Humans
  • Immunoglobulin Fab Fragments / immunology
  • Protein Binding / genetics
  • Receptors, Tumor Necrosis Factor / immunology*
  • Small Molecule Libraries


  • Complementarity Determining Regions
  • Immunoglobulin Fab Fragments
  • Receptors, Tumor Necrosis Factor
  • Small Molecule Libraries