Probing a protein–protein interaction by in vitro evolution
| Autor: | E. Suzanne Cohen, Phill Monk, Alexis C. Cockroft, Ralph Minter, Andrew Buchanan, Lutz Jermutus, Cathy Joberty Candotti, Celia P. Shorrock-Hart, George Thom, David Lowne |
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| Rok vydání: | 2006 |
| Předmět: |
Models
Molecular Phage display Protein Conformation Antibody Affinity Immunoglobulin Variable Region Mutagenesis (molecular biology technique) Computational biology Biology medicine.disease_cause Antibodies Cell Line Protein structure Peptide Library medicine Humans Amino Acid Sequence Genetics Mutation Interleukin-13 Multidisciplinary Biological Sciences Alanine scanning Complementarity Determining Regions Mutagenesis Immunoglobulin G Ribosome display Paratope Directed Molecular Evolution Ribosomes Systematic evolution of ligands by exponential enrichment |
| Zdroj: | Proceedings of the National Academy of Sciences. 103:7619-7624 |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | In this study, we used in vitro protein evolution with ribosome and phage display to optimize the affinity of a human IL-13-neutralizing antibody, a therapeutic candidate for the treatment of asthma, >150-fold to 81 pM by using affinity-driven stringency selections. Simultaneously, the antibody potency to inhibit IL-13-dependent proliferation in a cell-based functional assay increased 345-fold to an IC 50 of 229 pM. The panoply of different optimized sequences resulting from complementarity-determining region-targeted mutagenesis and error-prone PCR using ribosome display was contrasted with that of complementarity-determining region-targeted mutagenesis alone using phage display. The data highlight the advantage of the ribosome-display approach in identifying beneficial mutations across the entire sequence space. A comparison of mutation hotspots from in vitro protein evolution to knockout mutations from alanine scanning demonstrated that in vitro evolution selects the most appropriate positions for improvements in potency without mutating any of the key residues within the functional paratope. |
| Databáze: | OpenAIRE |
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