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Directed evolution towards protease-resistant hirudin variants.

Frank Wirsching1, Martina Keller, Christian Hildmann

  • 1Abteilung fuer Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Grisebachstr. 8, 37077, Goettingen, Germany.

Molecular Genetics and Metabolism
|December 5, 2003
PubMed
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Recombinant hirudin variants resistant to proteases were created, but protease resistance reduced thrombin inhibition. Proline substitutions offer a strategy for designing protease-resistant polypeptides.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Hirudin is a thrombin-specific inhibitor.
  • Hirudin is susceptible to digestion by proteases like pepsin and chymotrypsin.
  • Developing protease-resistant hirudin variants is crucial for therapeutic applications.

Purpose of the Study:

  • To generate and characterize recombinant hirudin variants with enhanced protease resistance.
  • To investigate the trade-off between protease resistance and thrombin inhibition activity.
  • To explore proline substitutions as a strategy for designing protease-resistant polypeptides.

Main Methods:

  • Phage display selection and high-throughput screening were used to generate hirudin variants.
  • Protease susceptibility assays were performed to assess resistance to pepsin and chymotrypsin.

Related Experiment Videos

  • Thrombin inhibition activity was measured using IC50 values.

    • Main Results:

    • Few hirudin variants with substitutions in the amino-terminal domain and carboxyl-terminal tail retained thrombin inhibition activity.
    • Mutations conferring protease resistance generally diminished thrombin inhibition activity.
    • A variant with proline substitutions (Pro50, Pro62, Pro63) showed complete resistance to pepsin and chymotrypsin but a 100-fold increase in IC50.

    Conclusions:

    • Protease resistance and thrombin inhibition activity are inversely correlated in hirudin variants.
    • Proline substitutions at major protease cleavage sites can confer broad protease resistance.
    • Proline substitutions represent a viable strategy for engineering protease-resistant polypeptides.