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Click display: a rapid and efficient in vitro protein display method for directed evolution.

Yu Zeng1, Michael Woolley1, Karuppiah Chockalingam1

  • 1Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, Bryan, TX 77807, USA.

Nucleic Acids Research
|August 7, 2023
PubMed
Summary

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This summary is machine-generated.

Click display is a novel in vitro protein display method that creates stable protein-cDNA complexes quickly. This technique enables efficient protein binder discovery and engineering, even in challenging environments.

Area of Science:

  • Biotechnology
  • Molecular Biology
  • Synthetic Biology

Background:

  • Traditional in vitro protein display methods often require intact RNA, limiting their application.
  • RNase-rich environments pose challenges for maintaining RNA integrity during biopanning.

Purpose of the Study:

  • To introduce a novel in vitro protein display method, termed click display.
  • To demonstrate the efficiency and robustness of click display for protein binder discovery and engineering.

Main Methods:

  • Developed a one-pot system integrating transcription, translation, and reverse transcription.
  • Utilized a modified DNA linker (ML) formed via click chemistry for stable protein-cDNA linkage.
  • Employed biopanning of a click-displayed library coupled with next-generation sequencing.

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Main Results:

  • Achieved covalent protein-cDNA complex formation within 2 hours.
  • Demonstrated >600-fold enrichment of target binders in a single round of biopanning.
  • Generated a library of Designed Ankyrin Repeat Proteins (DARPins) and identified nanomolar binders after six rounds of panning.

Conclusions:

  • Click display is a powerful and convenient tool for in vitro protein binder discovery and engineering.
  • The method is effective even in RNase-rich environments, expanding its applicability.
  • Facilitates rapid selection and optimization of protein binders.