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Profiling Protease Specificity: Combining Yeast ER Sequestration Screening (YESS) with Next Generation Sequencing.

Qing Li1, Li Yi1, Kam Hon Hoi1

  • 1Department of Chemistry, ‡Department of Biomedical Engineering, §Department of Chemical Engineering, and ∥Section of Molecular Genetics and Microbiology, University of Texas , Austin, Texas 78712, United States.

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|December 16, 2016
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Summary

A new Yeast Endoplasmic Reticulum (ER) Sequestration Screening (YESS) method combined with Next-Generation Sequencing (NGS) comprehensively maps protease specificity. This reveals an endogenous Kex2 protease cleavage pattern in yeast, crucial for yeast display technology users.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzyme Engineering

Background:

  • Yeast Endoplasmic Reticulum (ER) Sequestration Screening (YESS) is an enzyme engineering technology.
  • Profiling protease specificity is essential for protein engineering and biopharmaceutical development.

Purpose of the Study:

  • To establish a novel method combining YESS with Next-Generation Sequencing (NGS) for comprehensive protease specificity profiling.
  • To map in vivo proteolysis within the yeast secretory pathway.
  • To identify endogenous yeast proteases that may affect protein display technologies.

Main Methods:

  • A combinatorial substrate library was targeted to the yeast ER and processed through the secretory pathway.
  • Multicolor Fluorescence-Activated Cell Sorting (FACS) was used to isolate cells based on antibody labeling.
  • Next-Generation Sequencing (NGS) was employed to analyze cleaved substrates and determine protease specificity.
  • Kex2 knockout strains and in vitro assays were used to verify protease activity.

Main Results:

  • The YESS-NGS method successfully profiled the specificity of tobacco etch mosaic virus protease (wild-type and engineered).
  • A major in vivo cleavage pattern (Ali/Leu-X-Lys/Arg-Arg) was identified in the yeast secretory pathway.
  • This cleavage pattern was attributed to the endogenous protease Kex2.
  • The findings highlight potential Kex2-mediated removal of library members in yeast display screens.

Conclusions:

  • The YESS-NGS platform provides a powerful tool for comprehensive protease specificity profiling.
  • Understanding endogenous protease activity, like Kex2, is critical for optimizing yeast-based protein display and engineering systems.
  • Researchers using yeast display should be aware of potential Kex2 cleavage impacting their library selection.