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Directed in Vitro Evolution and Crystallographic Analysis of a Peptide-binding Single Chain Antibody Fragment (scFv) with Low Picomolar Affinity
- Source :
- Journal of Biological Chemistry. 279:18870-18877
- Publication Year :
- 2004
- Publisher :
- Elsevier BV, 2004.
-
Abstract
- We generated a single chain Fv fragment of an antibody (scFv) with a binding affinity of about 5 pm to a short peptide by applying rigorous directed evolution. Starting from a high affinity peptide binder, originally obtained by ribosome display from a murine library, we generated libraries of mutants with error-prone PCR and DNA shuffling and applied off-rate selection by using ribosome display. Crystallographic analysis of the scFv in its antigen-bound and free state showed that only few mutations, which do not make direct contact to the antigen, lead to a 500-fold affinity improvement over its potential germ line precursor. These results suggest that the affinity optimization of very high affinity binders is achieved by modulating existing interactions via subtle changes in the framework rather than by introducing new contacts. Off-rate selection in combination with ribosome display can evolve binders to the low picomolar affinity range even for peptide targets.
- Subjects :
- Models, Molecular
Static Electricity
Immunoglobulin Variable Region
Peptide binding
Peptide
Biology
Crystallography, X-Ray
Biochemistry
Mice
Peptide Library
Animals
Binding site
Peptide library
Molecular Biology
chemistry.chemical_classification
Binding Sites
Cell Biology
Directed evolution
DNA shuffling
Crystallography
Amino Acid Substitution
chemistry
Mutation
Ribosome display
Directed Molecular Evolution
Peptides
Systematic evolution of ligands by exponential enrichment
Protein Binding
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 279
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....b4c8a407986b7bf9b884ddccfd8b6194