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Characterizing Protein-Protein Interactions Using Deep Sequencing Coupled to Yeast Surface Display.

Angelica V Medina-Cucurella1, Timothy A Whitehead2,3

  • 1Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI, USA.

Methods in Molecular Biology (Clifton, N.J.)
|April 2, 2018
PubMed
Summary

We developed a method to analyze protein binder mutants, revealing sequence-function relationships and fine conformational epitopes. This technique combines deep sequencing, mutagenesis, and cell sorting for comprehensive analysis.

Keywords:
Conformational epitope mappingDeep sequencingFACSNicking mutagenesisYeast surface display

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Understanding protein-protein interactions is crucial for biological research and drug development.
  • Characterizing the impact of single amino acid changes (mutants) on protein binder function is essential for optimizing therapeutic proteins.
  • Existing methods may not provide comprehensive analysis of all possible single-point mutants.

Purpose of the Study:

  • To present a novel method for determining the affinity and specificity of nearly all single-point mutants for a full-length protein binder.
  • To enable the study of sequence-function relationships in protein-protein interactions.
  • To facilitate the determination of fine conformational epitopes on protein binders.

Main Methods:

  • The method integrates comprehensive mutagenesis to generate all single-point mutants.
  • Yeast surface display is employed to present these mutants on the cell surface.
  • Deep sequencing and fluorescence-activated cell sorting (FACS) are used to quantify the binding affinity and specificity of each mutant.

Main Results:

  • The described approach allows for the high-throughput characterization of thousands of protein mutants.
  • It provides quantitative data on how individual mutations affect protein binder affinity and specificity.
  • The data generated can map critical residues involved in protein-protein interactions and define the epitope.

Conclusions:

  • This combined methodology offers a powerful tool for dissecting sequence-function relationships in protein binders.
  • It enables a detailed understanding of the conformational epitope at a fine-grained level.
  • The approach is broadly applicable to various protein binders and interaction studies.