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Related Experiment Video

Updated: Aug 27, 2025

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators
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Reprogramming the Cleavage Specificity of Botulinum Neurotoxin Serotype B1.

Joseph D Cleveland1, Amir Taslimi1, Qi Liu1

  • 1Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado 80045 United States.

ACS Synthetic Biology
|September 26, 2022
PubMed
Summary

Researchers engineered an orthogonal botulinum neurotoxin serotype B (BoNT/B) protease for synthetic biology. This novel protease system enables precise control of cellular functions and facilitates the discovery of new protease-substrate pairs.

Keywords:
botulinum neurotoxincleavage reporterorthogonalproteasesubstratetranscriptional circuityeast screen

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Area of Science:

  • Synthetic biology
  • Molecular biology
  • Biochemistry

Background:

  • Reprogramming protease specificity is crucial for synthetic biology and biomedicine.
  • Engineering orthogonal proteases with high specificity for novel targets remains a significant challenge.

Purpose of the Study:

  • To engineer an orthogonal botulinum neurotoxin serotype B (BoNT/B) protease system with a nonnative substrate.
  • To demonstrate the utility of this system for regulating transcriptional circuitry and for in vivo protease evolution.

Main Methods:

  • Designed and validated an orthogonal BoNT/B protease and substrate in mammalian cells.
  • Incorporated mutations in protease and substrate, truncated target sequences, and cleavage reporters.
  • Transposed the platform to yeast for high-throughput screening and in vivo evolution.

Main Results:

  • Successfully engineered an orthogonal BoNT/B protease-substrate system in mammalian cells.
  • Demonstrated its application in controlling complex transcriptional circuitry.
  • Utilized the platform for high-throughput screening to identify novel orthogonal protease-substrate combinations in yeast.

Conclusions:

  • The developed orthogonal BoNT/B protease system expands protease versatility for synthetic biology applications.
  • This platform facilitates the discovery of new reprogrammed proteases and cleavage reporters.
  • The approach holds promise for advancing botulinum protease applications in diverse biological contexts.