Protein Engineering, Vol. 12, No. 4, 349-356,
April 1999
© 1999 Oxford University Press
In vitro scanning saturation mutagenesis of all the specificity determining residues in an antibody binding site
1 Department of Chemistry and Biochemistry, 2 Institute for Cellularand Molecular Biology and 3 Department of Chemical Engineering,The University of Texas at Austin, Austin, TX 78712, USA
For the first time, each specificity determining residue (SDR) in the binding site of an antibody has been replaced with every other possible single amino acid substitution, and the resulting mutants analyzed for binding affinity and specificity. The studies were conducted on a variant of the 26-10 antidigoxin single chain Fv (scFv) using in vitro scanning saturation mutagenesis, a new process that allows the high throughput production and characterization of antibody mutants [Burks,E.A., Chen,G., Georgiou,G. and Iverson,B.L. (1997) Proc. Natl Acad. Sci. USA, 94, 412–417]. Single amino acid mutants of 26-10 scFv were identified that modulated specificity in dramatic fashion. The overall plasticity of the antibody binding site with respect to amino acid replacement was also evaluated, revealing that 86% of all mutants retained measurable binding activity. Finally, by analyzing the physical properties of amino acid substitutions with respect to their effect on hapten binding, conclusions were drawn regarding the functional role played by the wild-type residue at each SDR position. The reported results highlight the value of in vitro scanning saturation mutagenesis for engineering antibody binding specificity, for evaluating the plasticity of proteins, and for comprehensive structure–function studies and analysis.
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