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An efficient method for generating proteins with altered enzymatic properties: application to beta-lactamase.

A R Oliphant1, K Struhl

  • 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.

Proceedings of the National Academy of Sciences of the United States of America
|December 1, 1989
PubMed
Summary

Researchers created altered beta-lactamase proteins using degenerate oligonucleotides to modify enzyme active sites. This method successfully generated enzymes with new antibiotic resistance profiles and altered functions.

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Area of Science:

  • Protein Engineering
  • Molecular Biology
  • Enzyme Function Analysis

Background:

  • Degenerate oligonucleotides are valuable tools for identifying essential sequences in proteins and nucleic acids.
  • Replacing DNA segments with these oligonucleotides allows for functional sequence discovery via genetic or biochemical selection.

Purpose of the Study:

  • To generate a diverse library of altered beta-lactamase proteins by modifying the active site.
  • To identify enzymes with novel properties, including altered substrate specificity and resistance to inhibitors.

Main Methods:

  • A library of 500,000 altered beta-lactamase proteins was created by inserting a mixed-base oligonucleotide into the gene sequence.
  • Functional screening identified approximately 2000 enzymes conferring ampicillin resistance in Escherichia coli.

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  • Fifty-eight selected enzymes were further characterized using various beta-lactam substrates.

    • Main Results:

    • Altered beta-lactamases exhibited modified specificity against different antibiotics.
    • Enzymes displayed resistance to suicide inhibitors like clavulanic acid and sulbactam.
    • Temperature-dependent activities and the responsible amino acid residues were identified.

    Conclusions:

    • This oligonucleotide-based approach is an effective and general method for creating proteins with novel properties.
    • The technique is particularly useful for protein engineering when high-resolution structural data is unavailable.