Rapid mapping of protein binding sites and conformational epitopes by coupling yeast surface display to chemical labeling and deep sequencing

Najar, TA and Khare, S and Varadarajan, R (2018) Rapid mapping of protein binding sites and conformational epitopes by coupling yeast surface display to chemical labeling and deep sequencing. [Book Chapter]

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Abstract

Delineating the precise regions on an antigen that are targeted by antibodies is important for the development of vaccines and antibody therapeutics. X-ray crystallography and NMR are considered the gold standard for providing precise information about these binding sites at atomic resolution. However, these are labor-intensive and require purified protein at high concentration. We have recently described 1 a rapid and reliable method that overcomes these constraints, using a panel of single cysteine mutants of the protein of interest and now provide protocols to facilitate its adoption. Mutants are displayed on the yeast cell surface either individually or as a pool, and labeled covalently with a cysteine specific probe. Binding site residues are inferred by monitoring loss of ligand or antibody binding by flow cytometry coupled to deep sequencing of sorted populations, or Sanger sequencing of individual clones. Buried cysteine residues are not labeled and library sizes are small, facilitating rapid identification of binding-site residues. The methodology was used to identify epitopes on the bacterial toxin CcdB targeted by twenty-four different monoclonal antibodies as well as by polyclonal sera. The method does not require purified protein or protein structural information and can be applied to a variety of display formats.

Item Type:	Book Chapter
Publication:	Methods in Molecular Biology
Series.:	Methods in Molecular Biology
Publisher:	Humana Press Inc.
Additional Information:	The copyright for this article belongs to the Springer Science+Business Media, LLC, part of Springer Nature
Keywords:	cysteine; epitope; monoclonal antibody; epitope; protein binding, antibody combining site; antigen binding; cell surface; cell surface display; chemical labeling; conformation; epitope mapping; flow cytometry; ligand binding; nonhuman; Saccharomyces cerevisiae; Sanger sequencing; yeast cell; binding site; chemistry; epitope mapping; genetics; high throughput sequencing; human; immunology; peptide library; procedures; protein conformation; staining, Antibodies, Monoclonal; Binding Sites; Epitope Mapping; Epitopes; High-Throughput Nucleotide Sequencing; Humans; Peptide Library; Protein Binding; Protein Conformation; Saccharomyces cerevisiae; Staining and Labeling
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	24 Aug 2022 04:56
Last Modified:	24 Aug 2022 04:56
URI:	https://eprints.iisc.ac.in/id/eprint/76077

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