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Related Experiment Videos

Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage.

Eeva-Christine Brockmann1, Urpo Lamminmäki, Petri Saviranta

  • 1Department of Biotechnology, University of Turku, Tykistökatu 6A 6, FIN-20520 Turku, Finland. eechbr@utu.fi

Biochimica Et Biophysica Acta
|May 17, 2005
PubMed
Summary

Protein engineering enabled the display of dihydropteroate synthetase (DHPS) on M13 phage. A key mutation (Asp96Asn) significantly enhanced DHPS phage display efficiency, aiding protein engineering efforts.

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Area of Science:

  • Protein Engineering
  • Molecular Biology
  • Biotechnology

Background:

  • Phage display is a powerful selection technique in protein engineering.
  • Expressing cytoplasmic proteins, like dihydropteroate synthetase (DHPS), on phage can be challenging due to cellular localization and potential misfolding.
  • Overcoming these challenges is crucial for expanding the utility of phage display technology.

Purpose of the Study:

  • To engineer the cytoplasmic enzyme dihydropteroate synthetase (DHPS) for efficient display on M13 phage.
  • To identify specific mutations that enhance DHPS expression and stability in the phage display system.
  • To improve the overall efficiency of DHPS-based phage display for protein engineering applications.

Main Methods:

  • Protein engineering of DHPS, including cysteine residue replacement and screening for periplasmic expression.

Related Experiment Videos

  • Random mutagenesis of DHPS to introduce diversity.
  • Phage display selection using a conformation-specific anti-DHPS antibody over multiple rounds.
  • Analysis of selected clones to identify beneficial mutations and their synergistic effects.

    • Main Results:

    • Cysteine replacement alone improved DHPS phage display 12-fold.
    • Random mutagenesis and three rounds of selection increased library display efficiency 280-fold.
    • A common Asp96Asn mutation was identified as a major contributor to efficient DHPS phage display, acting synergistically with cysteine replacements.

    Conclusions:

    • The engineered DHPS exhibits significantly improved phage display efficiency.
    • The Asp96Asn mutation, in combination with cysteine replacements, is critical for successful DHPS display on M13 phage.
    • This strategy enhances the application of phage display for engineering challenging proteins like DHPS.