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Whole plasmid mutagenic PCR for directed protein evolution.

Ichiro Matsumura1, Lori A Rowe

  • 1Department of Biochemistry, Center for Fundamental and Molecular Evolution, Emory University School of Medicine, Rollins Research Center, Room 4119, 1510 Clifton Road, Atlanta, GA 30322, USA. imatsum@emory.edu

Biomolecular Engineering
|April 29, 2005
PubMed
Summary

Directed evolution enhanced Escherichia coli lacZ gene performance. Whole plasmid mutagenesis and screening yielded a beta-galactosidase variant with 100-fold improved catalytic efficiency, demonstrating a powerful protein engineering approach.

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Area of Science:

  • Molecular Biology
  • Protein Engineering
  • Enzyme Kinetics

Background:

  • Directed evolution is a key strategy for protein engineering.
  • Optimizing protein expression is crucial for high-throughput assays.
  • The Escherichia coli lacZ gene encoding beta-galactosidase is a widely used reporter system.

Purpose of the Study:

  • To enhance the performance of the Escherichia coli lacZ gene through directed evolution.
  • To develop a more sensitive and precise reporter system for high-throughput screening.
  • To demonstrate the utility of whole plasmid mutagenic PCR for protein evolution.

Main Methods:

  • Two rounds of directed evolution were employed.
  • The first round involved whole plasmid PCR and self-ligation to randomize the lacZ promoter, followed by in vivo genetic selection for a constitutive expression vector.

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  • The second round used mutagenic long PCR on the entire plasmid, followed by restriction enzyme digestion, T4 DNA ligase self-ligation, and transformation into E. coli.

    • Main Results:

    • A library of beta-galactosidase mutants was generated, with approximately 80% being hypomorphs, indicating an appropriate mutation rate.
    • Fourteen variants with increased activity using 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) were isolated.
    • A purified protein variant showed a 100-fold improvement in catalytic efficiency (kcat) with para-nitrophenyl-beta-d-galactopyranoside (pNP-gal) compared to the parent enzyme.

    Conclusions:

    • Directed evolution using whole plasmid mutagenic PCR is effective for improving protein function.
    • The optimized lacZ gene provides a significantly enhanced reporter system.
    • This methodology is valuable for advancing protein engineering and assay development.