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Antibiotic Selection00:57

Antibiotic Selection

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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
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PROFICS: A bacterial selection system for directed evolution of proteases.

Christina Kröß1, Petra Engele1, Bernhard Sprenger1

  • 1acib GmbH, Graz, Austria; Institute of Biochemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria.

The Journal of Biological Chemistry
|August 21, 2021
PubMed
Summary

We developed PROFICS, a bacterial system to rapidly engineer proteases for biotechnology and medicine. This method enhances protease activity and specificity, enabling new therapeutic and diagnostic applications.

Keywords:
E. colibiotechnologycaspasecircular permutationdirected evolutionenzyme mutationtag cleavageviral protease

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Area of Science:

  • Biochemistry and Molecular Biology
  • Protein Engineering
  • Biotechnology

Background:

  • Proteases are crucial in biotechnology, medicine, and as drug targets.
  • Efficient methods for engineering protease properties are highly sought after.
  • Current methods can be time-consuming and lack broad applicability.

Purpose of the Study:

  • To establish a novel bacterial selection system, PROFICS, for protease optimization.
  • To enable rapid engineering of proteases for biotechnology, therapeutics, and diagnostics.
  • To demonstrate PROFICS's utility in improving protease activity and specificity.

Main Methods:

  • Developed PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial system.
  • Utilized a reporter enzyme and an Escherichia coli knockout strain for selection.
  • Generated a toolbox to select for proteases with varied P1' residue preferences.

Main Results:

  • PROFICS enables overnight optimization of proteases.
  • Demonstrated functionality with viral proteases and human caspase-2.
  • Achieved up to a 25-fold increase in caspase-2 activity and improved P1' tolerance.

Conclusions:

  • PROFICS is an efficient, overnight bacterial selection system for protease engineering.
  • The system can enhance protease activity, specificity, and tolerance.
  • PROFICS offers versatile applications including drug resistance screening and specificity reprogramming.