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Related Experiment Videos

A method for directed evolution and functional cloning of enzymes

H Pedersen1, S Hölder, D P Sutherlin

  • 1Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA.

Proceedings of the National Academy of Sciences of the United States of America
|September 2, 1998
PubMed
Summary

This study presents a novel in vitro evolution system for protein catalysts using filamentous phage. This method enables efficient selection of active enzymes from mutant libraries, facilitating functional cloning of novel biocatalysts.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Developing efficient methods for protein catalyst evolution is crucial for biocatalysis.
  • Current methods often rely on sequence or structural homology, limiting the discovery of novel enzymes.
  • In vitro evolution systems offer a powerful approach to discovering enzymes with desired catalytic functions.

Purpose of the Study:

  • To describe a general scheme for in vitro evolution of protein catalysts in a biologically amplifiable system.
  • To develop a method for selecting active catalysts based on intramolecular substrate conversion.
  • To demonstrate the utility of this methodology using staphylococcal nuclease as a model.

Main Methods:

  • Utilizing filamentous phage displaying both the catalyst and a tethered substrate.

Related Experiment Videos

  • Employing site-specific covalent attachment of substrate to the phage pIII coat protein.
  • Implementing selection based on catalyst activity, either through release or attachment to a solid support.

    • Main Results:

    • Demonstrated a 100-fold enrichment of active staphylococcal nuclease displaying phage in a single step.
    • Analyzed factors influencing selection efficiency in the phage display system.
    • Showcased the ability to select active catalysts from a library of noncatalytic proteins.

    Conclusions:

    • The described in vitro evolution system is effective for selecting protein catalysts.
    • This methodology facilitates the functional cloning of natural enzymes based on catalytic activity.
    • The approach holds promise for discovering enzymes with diverse catalytic functions, such as glycosyl transfer and proteolysis.