Isolation of anti-T cell receptor scFv mutants by yeast surface display
| Autor: | David M. Kranz, Eric T. Boder, Michele C. Kieke, Bryan K. Cho, K D Wittrup |
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| Rok vydání: | 1997 |
| Předmět: |
Receptors
Antigen T-Cell alpha-beta Molecular Sequence Data Mutant Antibody Affinity Immunoglobulin Variable Region Bioengineering Saccharomyces cerevisiae Yeast display Biochemistry Epitope Flow cytometry Proto-Oncogene Proteins c-myc Epitopes medicine Biotinylation Genomic library Cloning Molecular Immunoglobulin Fragments Molecular Biology Fluorescent Dyes Gene Library Base Sequence medicine.diagnostic_test Chemistry Antibodies Monoclonal Flow Cytometry Molecular biology Yeast Subcloning Mutagenesis Plasmids Biotechnology |
| Zdroj: | Protein Engineering Design and Selection. 10:1303-1310 |
| ISSN: | 1741-0134 1741-0126 |
| DOI: | 10.1093/protein/10.11.1303 |
| Popis: | Yeast surface display and sorting by flow cytometry have been used to isolate mutants of an scFv that is specific for the Vbeta8 region of the T cell receptor. Selection was based on equilibrium binding by two fluorescently labeled probes, a soluble Vbeta8 domain and an antibody to the c-myc epitope tag present at the carboxy-terminus of the scFv. The mutants that were selected in this screen included a scFv with threefold increased affinity for the Vbeta8 and scFv clones that were bound with reduced affinities by the anti-c-myc antibody. The latter finding indicates that the yeast display system may be used to map conformational epitopes, which cannot be revealed by standard peptide screens. Equilibrium antigen binding constants were estimated within the surface display format, allowing screening of isolated mutants without necessitating subcloning and soluble expression. Only a relatively small library of yeast cells (3 x 10[5]) displaying randomly mutagenized scFv was screened to identify these mutants, indicating that this system will provide a powerful tool for engineering the binding properties of eucaryotic secreted and cell surface proteins. |
| Databáze: | OpenAIRE |
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