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A flow cytometry-based screening system for directed evolution of proteases.

Ran Tu1, Ronny Martinez, Radivoje Prodanovic

  • 1School of Engineering and Science, Jacobs University Bremen (JUB), Bremen, Germany.

Journal of Biomolecular Screening
|February 22, 2011
PubMed
Summary
This summary is machine-generated.

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A new flow cytometry method enables high-throughput screening of protease variants for improved industrial applications. This enzyme evolution technique successfully identified a protease with enhanced resistance to an inhibitor.

Area of Science:

  • Enzyme engineering
  • Biotechnology
  • Protein science

Background:

  • Proteases are vital industrial enzymes requiring enhanced catalytic efficiency and stability.
  • Existing directed evolution methods have limitations for high mutational loads.
  • Flow cytometry screening offers ultra-high throughput for enzyme variant selection.

Purpose of the Study:

  • To develop and validate a flow cytometry-based screening system for directed evolution of proteases.
  • To enable high-throughput screening of protease variants under high mutational loads.
  • To identify protease variants with improved inhibitor resistance.

Main Methods:

  • Utilized a protease-deficient Bacillus subtilis strain (WB800N) and a model protease (subtilisin Carlsberg).

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  • Employed water-in-oil-in-water double emulsion technology for compartmentalization.
  • Encapsulated B. subtilis cells with a fluorogenic substrate for protease activity detection.
  • Screened an epPCR mutant library for increased resistance to antipain dihydrochloride.

    • Main Results:

    • Successfully developed a flow cytometry screening system for protease directed evolution.
    • Validated the system by screening a high-mutational-load library.
    • Isolated a protease variant (K127R, T237P, M239I, I269V, Y310F, I372V) with improved antipain dihydrochloride resistance.

    Conclusions:

    • The developed flow cytometry screening technology is effective for directed protease evolution.
    • This method facilitates the identification of protease variants with enhanced inhibitor resistance.
    • The technology opens new opportunities for enzyme engineering of proteases for industrial applications.