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Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells
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A protein-fragment complementation assay to quantify synthetic protein scaffold efficiency.

Pascale Lemieux1,2,3,4, Alexandre K Dubé1,2,3,4, Christian R Landry1,2,3,4,5,6

  • 1Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Québec G1V 0A6, QC, Canada.

Synthetic Biology (Oxford, England)
|March 16, 2026
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Summary

Protein scaffolds enhance synthetic biology by bringing enzymes together. Researchers used a novel assay in yeast to find new scaffold components, improving protein scaffold design for biotechnology applications.

Keywords:
DHFR PCAPDZ domainSH3 domainsynthetic protein scaffold

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Area of Science:

  • Synthetic biology
  • Biotechnology
  • Protein engineering

Background:

  • Protein scaffolds are crucial tools in synthetic biology, enabling applications like increased yield and optimized signaling specificity.
  • Current PBD-peptide combinations for scaffolds in yeast have shown limited success, necessitating further exploration.
  • Characterizing scaffold efficiency is challenging due to difficulties in quantitatively measuring pathway outputs.

Purpose of the Study:

  • To explore and characterize novel peptide-binding domain (PBD)-peptide pairs for protein scaffold design in yeast.
  • To establish a robust method for quantifying scaffold efficiency using the dihydrofolate reductase protein-fragment complementation assay (DHFR PCA).
  • To identify key factors influencing scaffold performance, including PBD-peptide interaction strength, binding availability, scaffold architecture, and expression levels.

Main Methods:

  • Utilized the DHFR PCA in yeast to couple scaffold efficiency with cell growth rate.
  • Characterized the strength of PBD-peptide interactions (PPI) and the in vivo binding availability of PBDs and peptides.
  • Tested various scaffold architectures and expression levels to assess their impact on scaffolding efficiency.
  • Employed DHFR PCA to screen and identify novel PBD-peptide pairs.

Main Results:

  • Scaffold efficiency is critically dependent on the PPI strength of the weakest binding PBD-peptide pair.
  • In vivo binding availability of certain domains and peptides limits overall PPI strength.
  • Minor changes in scaffold architecture and expression levels significantly affect scaffolding efficiency as detected by DHFR PCA.
  • Identified novel PBD-peptide pairs suitable for expanding the toolbox for yeast scaffold design.

Conclusions:

  • The DHFR PCA provides an effective and quantitative method for characterizing protein scaffold efficiency in yeast.
  • Optimizing PPI strength and ensuring sufficient binding availability are key for effective protein scaffold design.
  • Novel PBD-peptide pairs have been identified, expanding options for engineering protein scaffolds in yeast.
  • This work facilitates the development of improved synthetic biology tools for biotechnological applications.