Protease engineering: Approaches, tools, and emerging trends.

Journal: Biotechnology advances
Published Date: Sep 1, 2025

Abstract

Engineered proteases with bespoke substrate specificities and activities can empower broad and innovative applications in biomedicine, mass spectrometry-based proteomics, and chemical and synthetic biology. This review provides an authoritative, topical, and detailed description and discussion of the directed evolution and high-throughput strategies designed to engineer the substrate specificity of proteases in E. coli, yeast, phage, and cell-free systems. Second, we discuss emerging protease engineering strategies that complement directed evolution, including antibody-protease fusions that enable proximity catalysis, and protease substrate specificity switching driven by exogenous protein-protein interactions. Lastly, we discuss principles for engineering split and autoinhibited proteases, which are key signal-processing modules in protein circuits. Overall, readers will gain a valuable understanding of the latest advances in protease engineering, focusing on methodologies and strategies that enable precise control of protease activity and specificity.

Authors

  • Samantha G Martinusen
    Department of Chemical Engineering, University of Florida, Gainesville 32611, USA.
  • Sage E Nelson
    Department of Chemical Engineering, University of Florida, Gainesville 32611, USA.
  • Ethan W Slaton
    Department of Chemical Engineering, University of Florida, Gainesville 32611, USA.
  • Lawton F Long
    Department of Chemical Engineering, University of Florida, Gainesville 32611, USA.
  • Raymond Pho
    Department of Chemical Engineering, University of Florida, Gainesville 32611, USA.
  • Seyednima Ajayebi
    Department of Chemical Engineering, University of Florida, Gainesville 32611, USA.
  • Carl A Denard
    Department of Chemical Engineering, University of Florida, Gainesville 32611, USA; UF Health Cancer Center, University of Florida, Gainesville, 32611, USA. Electronic address: cdenard@ufl.edu.

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