ArticleProceedings of the National Academy of Sciences of the United States of America1997
In vitro scanning saturation mutagenesis of an antibody binding pocket.
Article in Proceedings of the National Academy of Sciences of the United States of America, 1997. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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Who cites it
17 citing papers in PubMed.
- Engineering affinity-matured variants of an anti-polysialic acid monoclonal antibody with superior cytotoxicity-mediating potency.Cell chemical biology · 2025Article
- Immunosensor for Rapid and Sensitive Detection of Digoxin.ACS omega · 2023Article
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- A High-Throughput Method for Directed Evolution of NAD(P)ACS catalysis · 2019Article
- Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation.International journal of molecular sciences · 2019Review
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- Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding.Proceedings of the National Academy of Sciences of the United States of America · 2017Article
- One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections.PloS one · 2016Article
- Generation and purification of highly specific antibodies for detecting post-translationally modified proteins in vivo.Nature protocols · 2014Article
- Phage-displayed peptide that mimics aflatoxins and its application in immunoassay.Journal of agricultural and food chemistry · 2013Article
- Closely related antibody receptors exploit fundamentally different strategies for steroid recognition.Proceedings of the National Academy of Sciences of the United States of America · 2008Article
- Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3.Protein science : a publication of the Protein Society · 2006Article
- Steroid binding by antibodies and artificial receptors: exploration of theoretical methods to determine the origins of binding affinities and specificities.Journal of computer-aided molecular design · 2000Article
- Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity.Proceedings of the National Academy of Sciences of the United States of America · 2000Article
- In vitro analysis of roles of a disulfide bridge and a calcium binding site in activation of Pseudomonas sp. strain KWI-56 lipase.Journal of bacteriology · 2000Article
- Article
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4 authors.
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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.
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