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A molecular sensor system based on genetically engineered alkaline phosphatase

C A Brennan1, K Christianson, M A La Fleur

  • 1Abbott Laboratories, North Chicago, IL 60064-4000, USA.

Proceedings of the National Academy of Sciences of the United States of America
|June 20, 1995
PubMed
Summary

Engineered alkaline phosphatase (AP) hybrid proteins detect antibodies by modulating enzyme activity. Point mutations in AP alter antibody binding effects, switching from inhibition to activation, demonstrating a general detection mechanism.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Immunotechnology

Background:

  • Escherichia coli alkaline phosphatase (AP) is a well-characterized enzyme.
  • Antibody detection systems often rely on enzyme-based signaling.
  • Modulating enzyme activity upon antibody binding offers a novel detection strategy.

Purpose of the Study:

  • To design and characterize novel binding and signaling proteins for antibody detection.
  • To investigate the effect of AP point mutations on antibody-mediated enzyme activity modulation.
  • To demonstrate the general applicability of this AP-based detection system.

Main Methods:

  • Construction of hybrid proteins by inserting peptide epitopes into wild-type and mutant AP.
  • Characterization of antibody binding to hybrid proteins and its effect on AP enzyme activity.

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  • Utilizing epitopes from human immunodeficiency virus type 1 gp120 and hepatitis C virus core protein.

    • Main Results:

    • Antibody binding to wild-type AP hybrids resulted in decreased enzyme activity (up to 40% inhibition).
    • Antibody binding to AP mutant hybrids resulted in increased enzyme activity (up to 300% activation).
    • This modulation trend was consistent across different epitopes and antibodies, indicating a general phenomenon.

    Conclusions:

    • AP-epitope hybrid proteins serve as effective antibody sensors.
    • Single amino acid changes in AP's active site can switch modulation from inhibition to activation.
    • This system offers a versatile and generalizable platform for antibody detection.