In vitroscanning saturation mutagenesis of an antibody binding pocket

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Abstract
We have combined PCR mutagenesis with in vitro transcription/translation and ELISA for the rapid generation and characterization of antibody mutants. The PCR products are used directly as the template for the in vitro transcription/translation reactions and because no cloning steps are required, the in vitro saturation mutagenesis of one residue can be completed in duplicate within a week by a single investigator. In vitro scanning saturation mutagenesis was used to analyze the role and plasticity of six key contact residues (H:Tyr-33, H:Asn-35, H:Tyr-50, H:Trp-100, L:Val-94, and L:Pro-96) in the binding pocket of a single chain Fv antibody derived from the 26–10 monoclonal antibody. A total of 114 mutant antibodies were produced; all 19 substitutions at each of the 6 chosen positions. The mutants were analyzed for binding to digoxin, digitoxin, digoxigenin, and ouabain resulting in the generation of a comprehensive data base of 456 relative affinity values. Excellent agreement between the relative affinity values obtained with in vitro synthesized mutant antibodies and equilibrium affinity data obtained with previously reported purified mutant monoclonal antibodies was observed. Approximately 75% of the single amino acid mutants exhibited significant binding to one or more of the digoxin analogs. Mutations that alter and, in some cases, reverse specificity for the different digoxin analogs were identified. In vitro scanning saturation mutagenesis represents a new tool for protein structure-function and engineering studies and can be interfaced with laboratory automation so that an even higher throughput of protein mutants can be constructed and analyzed.