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The Evidence for Evolution02:55

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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
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Directed Evolution of Split APEX2 Peroxidase.

Yisu Han1, Tess Caroline Branon1, Jeffrey D Martell1

  • 1Department of Chemistry , Massachusetts Institute of Technology , Cambridge , Massachusetts 02139 , United States.

ACS Chemical Biology
|March 9, 2019
PubMed
Summary
This summary is machine-generated.

Researchers engineered a split APEX (sAPEX) tool by evolving inactive enzyme fragments that regain peroxidase activity upon molecular interaction. This innovation expands applications in spatial proteomics and transcriptomics.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • APEX is an engineered peroxidase with diverse applications including subcellular staining and proximity biotinylation.
  • Advancing APEX capabilities is crucial for enhancing spatial proteomics and transcriptomics.

Purpose of the Study:

  • To engineer a split APEX (sAPEX) tool using directed evolution.
  • To enable reconstituted peroxidase activity through molecular interactions between APEX fragments.

Main Methods:

  • Directed evolution using yeast display and fluorescence-activated cell sorting (FACS).
  • Engineering of two inactive fragments, AP (N-terminal) and EX (C-terminal).
  • Demonstration of sAPEX reconstitution in various cellular compartments and contexts.

Main Results:

  • Developed a 200-amino-acid N-terminal fragment (AP) and a 50-amino-acid C-terminal fragment (EX).
  • AP and EX fragments individually showed no peroxidase activity.
  • Reconstitution of peroxidase activity was achieved when AP and EX were brought together by molecular interactions.

Conclusions:

  • sAPEX enables controllable peroxidase activity through fragment reconstitution.
  • sAPEX demonstrates successful application in mammalian cytosol, RNA scaffolds, and organelle contact sites.
  • This split enzyme system offers novel possibilities for spatial biology and chemical labeling.