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Related Experiment Videos

Switching antibody specificity through minimal mutation.

Andrea Piatesi1, Caroline Aldag, Donald Hilvert

  • 1Laboratory of Organic Chemistry, ETH Zürich, Hönggerberg HCI F 339, CH-8093 Zürich, Switzerland.

Journal of Molecular Biology
|February 26, 2008
PubMed
Summary
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Antibody engineering successfully reengineered an antibody to bind diverse steroids. Subtle mutations switched antibody selectivity, demonstrating facile creation of specific binding pockets.

Area of Science:

  • Biochemistry
  • Immunology
  • Protein Engineering

Background:

  • Antibody 1E9, initially generated against a hexachloronorbornene derivative, catalyzes Diels-Alder reactions.
  • Reengineering antibodies offers a pathway to create novel binding specificities.

Purpose of the Study:

  • To reengineer antibody 1E9 for binding structurally diverse steroids.
  • To investigate the impact of specific mutations on antibody binding affinity and selectivity.

Main Methods:

  • Antibody reengineering through targeted mutagenesis.
  • Steroid binding assays to determine affinity (K(d)) and specificity.
  • Comparison of wild-type antibody, single mutants, and double mutants.

Main Results:

Related Experiment Videos

  • Antibody 1E9 was successfully reengineered to bind various steroids with nanomolar affinities.
  • Two specific mutations (Leu(H47)Trp/Arg(H100)Trp) switched antibody selectivity to mimic the progesterone-binding antibody DB3.
  • Single mutants exhibited high specificity for 5alpha-pregnan-3beta-ol-20-one, outperforming DB3.
  • The double mutant bound multiple steroids with varying A-B ring configurations.

Conclusions:

  • Subtle amino acid changes can significantly alter antibody binding pockets and selectivity.
  • This demonstrates the potential for easily creating tailored binding specificities from a common antibody scaffold.
  • The findings provide insights into antibody structure-function relationships and protein engineering strategies.