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A gasdermin-based life-death evolution system for reprogramming protease specificity.

Ziqi Gao1, Tianzhen Li1, Hao Ye1

  • 1Department of Chemistry, Research Center for Chemical Biology and Omics Analysis, Guangdong Provincial Key Laboratory of Catalysis, College of Science, Southern University of Science and Technology, Shenzhen, China.

Nature Chemical Biology
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Summary
This summary is machine-generated.

Researchers developed a novel system for reprogramming protease specificity. This method uses a toxic protein marker to efficiently select for engineered proteases, enabling applications in proteome editing and therapeutics.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Synthetic Biology

Background:

  • Protease specificity reprogramming is crucial for applications like proteome editing and therapeutics.
  • Existing methods for protease engineering face challenges in scalability and efficiency.

Purpose of the Study:

  • To develop a novel in vivo life-death selection system for efficient protease specificity reprogramming.
  • To demonstrate the platform's ability to select and enrich protease variants with altered substrate specificities.

Main Methods:

  • A modular directed evolution system was designed using the toxic N-terminal domain of gasdermin D (GD-N) protein as a selection marker.
  • Bacterial cells expressing GD-N with an engineered cleavage site were subjected to selection, enriching for functional protease variants that render GD-N non-toxic.
  • The tobacco etch virus protease (TEVp) and its substrate were used as a model system to validate the platform's efficacy.

Main Results:

  • The selection system successfully enriched for efficient protease variants by over a millionfold in a single round of directed evolution.
  • Engineered TEVp variants demonstrated the ability to cleave specific sequences on target proteins with pathological relevance.
  • The platform demonstrated modularity, allowing for the exploration of vast protease mutational diversity.

Conclusions:

  • The developed in vivo life-death selection system provides a powerful and efficient platform for protease reprogramming.
  • This technology has significant potential for advancing proteome editing, therapeutic interventions, and the development of novel biochemical tools.