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Design, selection, and characterization of a split chorismate mutase.

Manuel M Müller1, Hajo Kries, Eva Csuhai

  • 1Laboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich, Switzerland.

Protein Science : a Publication of the Protein Society
|March 23, 2010
PubMed
Summary

Researchers engineered a small split enzyme from a thermostable chorismate mutase (CM). This engineered split CM (chorismate mutase) functions as a sensitive tool for protein-protein interaction studies and enzyme engineering.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Split proteins are crucial for studying protein-protein interactions and folding.
  • Enzymes engineered from split protein systems offer unique advantages for biological research.

Purpose of the Study:

  • To engineer a novel, small split enzyme based on thermostable chorismate mutase (CM).
  • To develop a split CM system for detecting protein-protein interactions and facilitating enzyme engineering.

Main Methods:

  • Dissecting a helical-bundle CM from Methanococcus jannaschii into two fragments.
  • Attaching leucine zipper dimerization domains to enzyme fragments.
  • Employing combinatorial mutagenesis and in vivo selection to optimize linker sequences.
  • Characterizing the assembled split enzyme activity.

Main Results:

  • A weakly active heterodimeric mutase was obtained after fragment assembly.
  • Optimized linker sequences led to spontaneous assembly of inactive fragments into an active split CM.
  • The engineered split CM exhibited wild-type like activity.

Conclusions:

  • The engineered split CM is a small, efficient tool for protein interaction detection.
  • Its simple topology and small size make it a valuable scaffold for enzyme engineering.
  • This system serves as a split sensor for specific protein-protein interactions.