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Directed Evolution Methods to Rewire Signaling Networks.

Raphaël B Di Roberto1, Benjamin M Scott1, Sergio G Peisajovich2

  • 1Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON, Canada, M5S 3G5.

Methods in Molecular Biology (Clifton, N.J.)
|March 16, 2017
PubMed
Summary
This summary is machine-generated.

Cells use complex signaling pathways to respond to their environment. New methods for creating and sorting mutant proteins help researchers study these intricate cellular networks and protein interactions.

Keywords:
Directed evolutionDomain shufflingError-prone PCRFluorescence-activated cell sortingProtein networkRandom mutagenesisSignaling pathway

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Area of Science:

  • Cellular Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Cellular responses to environmental stimuli rely on intricate signaling pathways.
  • These pathways involve complex networks of protein interactions crucial for cellular regulation.
  • Understanding protein activity within these native contexts is vital for biological studies and pathway engineering.

Purpose of the Study:

  • To present methods for generating mutant protein libraries with altered network interactions.
  • To detail a protocol for isolating these variants using fluorescence-activated cell sorting (FACS).
  • To exemplify these techniques using the G protein-coupled receptor Ste2 in Saccharomyces cerevisiae.

Main Methods:

  • Whole-gene point mutagenesis for generating mutant protein libraries.
  • Domain shuffling to create proteins with modified interaction capabilities.
  • Fluorescence-activated cell sorting for isolating functional variants in live cells.

Main Results:

  • Successful generation of mutant protein libraries exhibiting altered network interactions.
  • Demonstration of FACS for efficient isolation of specific variants.
  • Application of these methods to study G protein-coupled receptor Ste2 signaling in yeast.

Conclusions:

  • Advances in library construction and cell sorting facilitate the study of complex signaling networks.
  • These techniques enable deeper understanding of protein behavior in native cellular environments.
  • The presented methods offer powerful tools for research in cellular signaling and pathway engineering.