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Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing
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Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next

Maria Felicia Soluri1, Simone Puccio2, Giada Caredda2

  • 1Department of Health Sciences, Università del Piemonte Orientale & IRCAD, Novara, Italy.

Journal of Visualized Experiments : Jove
|October 23, 2018
PubMed
Summary

Researchers developed a "domainome" strategy using TEM-1 β-lactamase to select correctly folded protein domains. This method enables functional and structural studies, aiding gene annotation and antigen identification.

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Area of Science:

  • Molecular Biology
  • Protein Engineering
  • Genomics

Background:

  • Folding reporters are crucial tools for assessing protein folding and function.
  • Identifying functional protein domains is essential for understanding biological systems.
  • Current methods for domain discovery can be labor-intensive and limited in scope.

Purpose of the Study:

  • To develop a genomic-scale method for selecting correctly folded protein domains.
  • To create a "domainome" library suitable for structural and functional studies.
  • To demonstrate the utility of the "domainome" for selecting domains with specific binding properties.

Main Methods:

  • Utilizing TEM-1 β-lactamase as a folding reporter to select functional protein fragments from intronless genomes.
  • Cloning and displaying the "domainome" library in a phage display system.
  • Employing next-generation sequencing (NGS) for deep sequencing analysis of library diversity and selection outputs.

Main Results:

  • Successfully generated a "domainome" library of well-expressed and soluble protein fragments.
  • Demonstrated the selection of specific protein domains with desired binding properties (e.g., protein-protein or protein-antibody interactions).
  • Validated the use of deep sequencing for comprehensive analysis of selected fragments, including diversity, abundance, and mapping.

Conclusions:

  • The "domainome" strategy provides a powerful approach for discovering and characterizing functional protein domains.
  • This method facilitates structural and functional studies, aiding in gene annotation and antigen discovery.
  • The integration of phage display and NGS offers a robust platform for protein domain selection and analysis.