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Related Experiment Videos

Phage-assisted continuous evolution of proteases with altered substrate specificity.

Michael S Packer1,2,3, Holly A Rees1,3, David R Liu4,5,6

  • 1Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA, 02138, USA.

Nature Communications
|October 18, 2017
PubMed
Summary

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Researchers engineered a TEV protease using phage-assisted continuous evolution (PACE) to cleave a new target sequence in human IL-23. This modified protease inhibits IL-23 immune signaling, demonstrating custom protease development for therapeutic applications.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Proteases are enzymes that cleave peptide bonds, with specific sequences dictating their targets.
  • TEV protease canonically cleaves the ENLYFQS sequence.
  • Altering protease specificity is crucial for biotechnological and therapeutic applications.

Purpose of the Study:

  • To evolve TEV protease to cleave a novel target sequence (HPLVGHM) in human IL-23.
  • To characterize the substrate specificity and molecular basis of the evolved protease.
  • To establish a method for engineering custom proteases with altered specificities.

Main Methods:

  • Phage-assisted continuous evolution (PACE) was employed over approximately 2500 generations.
  • The evolved protease was tested for cleavage of human IL-23 and its effect on IL-23-mediated immune signaling in murine splenocytes.

Related Experiment Videos

  • Substrate specificity profiling and mutational dissection were used to characterize the enzyme.

    • Main Results:

    • A variant TEV protease with 20 mutations was evolved, capable of cleaving human IL-23 at the HPLVGHM site.
    • The evolved protease inhibited IL-23-mediated immune signaling in primary murine splenocyte cultures.
    • Specificity profiling revealed significant shifts and broadening at six altered amino acid positions.

    Conclusions:

    • PACE is effective for rapidly engineering proteases with altered substrate specificities.
    • The evolved protease demonstrates the potential for custom protease development to target specific proteins.
    • This work provides a foundation for developing proteases for biotechnological and therapeutic uses, including treating protease-deficiency diseases.