Abstract
In this chapter, we discuss a method to determine the affinity and specificity of nearly all single-point mutants for a full-length protein binder. This method combines deep sequencing, comprehensive mutagenesis, yeast surface display, and fluorescence-activated cell sorting. This approach has been used to study sequence-function relationships for protein-protein interactions. The data can be used to determine the fine conformational epitope on the protein binder.
Keywords:
Conformational epitope mapping; Deep sequencing; FACS; Nicking mutagenesis; Yeast surface display.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Binding Sites
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Cell Adhesion Molecules / chemistry
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Cell Adhesion Molecules / genetics
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Cell Adhesion Molecules / metabolism*
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Cell Separation
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Epitope Mapping / methods*
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Epitopes / chemistry*
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Epitopes / genetics
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Flow Cytometry
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High-Throughput Nucleotide Sequencing / methods*
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Mutagenesis
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Peptide Library
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Protein Binding
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Protein Interaction Domains and Motifs*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
Substances
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Cell Adhesion Molecules
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Epitopes
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Peptide Library
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Saccharomyces cerevisiae Proteins