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Related Experiment Videos

Improving a circularly permuted TEM-1 beta-lactamase by directed evolution.

Joel Osuna1, Alejandra Pérez-Blancas, Xavier Soberón

  • 1Instituto de Biotecnología, UNAM, Apdo. Postal 510-3 Cuernavaca, Morelos 62250, México. joel@ibt.unam.mx

Protein Engineering
|June 26, 2002
PubMed
Summary

Circular permutation of TEM beta-lactamase proteins was explored to understand protein structure. Directed evolution recovered enzyme activity, demonstrating protein engineering potential for novel enzyme variants.

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Area of Science:

  • Biochemistry
  • Protein Engineering
  • Molecular Biology

Background:

  • Protein structure is crucial for function.
  • Circular permutation is a technique to study protein folding and stability.
  • TEM beta-lactamase is an important enzyme for antibiotic resistance research.

Purpose of the Study:

  • To investigate the impact of circular permutation on TEM beta-lactamase structure and function.
  • To engineer improved TEM beta-lactamase variants through directed evolution.
  • To understand the role of secondary structure order in protein folding.

Main Methods:

  • Circular permutation of TEM beta-lactamase was created by introducing new N- and C-termini.
  • Directed evolution was employed using antibiotic resistance as a selection marker.

Related Experiment Videos

  • Mutants were generated using different connecting peptides and subjected to multiple evolution cycles.

    • Main Results:

    • The initial permuted TEM beta-lactamase showed significantly impaired activity.
    • Introduction of random connector peptides and directed evolution led to the isolation of active mutants.
    • A sixth-cycle variant with a GGS connector peptide exhibited 8% of the wild-type enzyme's catalytic efficiency.

    Conclusions:

    • Circularly permuted proteins can be engineered to regain or improve function.
    • Directed evolution is effective in selecting for beneficial mutations in permuted enzymes.
    • This study highlights the adaptability of protein structures and the potential for creating novel enzyme functionalities.