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Related Experiment Videos

Directed evolution studies with combinatorial libraries of T4 lysozyme mutants

P A Patten1, T Sonoda, M M Davis

  • 1Department of Chemistry, University of California at Berkeley 94720-1460, USA.

Molecular Diversity
|February 1, 1996
PubMed
Summary
This summary is machine-generated.

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Researchers explored protein evolution using T4 lysozyme, creating mutant gene libraries. While no new functions emerged, a novel E. coli gene was discovered that aids in detecting low-level enzymatic activity.

Area of Science:

  • Protein evolution
  • Enzymology
  • Molecular biology

Background:

  • Gene duplication and divergence drive protein evolution.
  • Quantifying new functions and mutational steps is experimentally challenging.
  • T4 lysozyme serves as a model system for studying protein adaptation.

Purpose of the Study:

  • To investigate the potential for new functions arising from T4 lysozyme.
  • To determine the mutational steps required for novel enzymatic activities.
  • To assess the diversity sampled by large mutant libraries.

Main Methods:

  • Synthesized two combinatorial libraries of T4 lysozyme mutant genes (>10^7 variants).
  • One library featured an average of 14 missense mutations; the other randomized 13 active site residues.

Related Experiment Videos

  • Selected mutants in E. coli strains deficient in beta-galactosidase (lacZ) or prephenate dehydratase (pheA) activity.

    • Main Results:

    • No T4 lysozyme mutants acquired beta-galactosidase or prephenate dehydratase activity.
    • A novel E. coli locus was identified that weakly complements these deficiencies.
    • This locus enabled colony formation, indicating a turnover number of ~1000 min^-1 (lacZ) or 25 min^-1 (pheA).

    Conclusions:

    • The study defines the limits of detectable evolved enzymatic activity in these selection systems.
    • Strong selective pressure revealed an unexpected compensatory mechanism.
    • Characterization of the novel E. coli locus will improve future selection experiments.