Expression of the Bacterial Enzyme IdeS Using a GFP Fusion in the Yeast Saccharomyces cerevisiae

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Abstract

Bacterial proteases are important enzymes used in several technical applications where controlled cleavage of proteins is needed. They are challenging enzymes to express recombinantly as parts of the proteome can be hydrolyzed by their activity. The eukaryotic model organism Saccharomyces cerevisiae is potentially a good expression host as it tolerates several stress conditions and is known to better express insoluble proteins compared to bacterial systems. In this chapter we describe how the protease IdeS from Streptococcus pyogenes can be expressed in S. cerevisiae. The expression of IdeS was followed by constructing a fused protein with GFP and measuring the fluorescence with flow cytometry. The protease presence was confirmed with a Western blot assay and activity was measured with an in vitro assay. To reduce potentially toxic effect on the host cell, the growth and production phases were separated by using the inducible promoter GAL1p to control recombinant gene expression. The protocol provided may be adopted for other bacterial proteases through minor modifications of the fused protein.

Original languageEnglish
Title of host publicationBacterial pathogenesis
Subtitle of host publicationMethods and protocols
EditorsPontus Nordenfelt, Mattias Collin
PublisherHumana Press
Chapter9
Pages131-146
Edition2nd
ISBN (Electronic)978-1-0716-3243-7
DOIs
Publication statusPublished - 2023

Publication series

NameMethods in molecular biology (Clifton, N.J.)
PublisherSpringer
ISSN (Print)1940-6029

Subject classification (UKÄ)

  • Microbiology in the medical area

Free keywords

  • Saccharomyces cerevisiae/genetics
  • Bacterial Proteins/metabolism
  • Recombinant Fusion Proteins/genetics
  • Fluorescence
  • Peptide Hydrolases/metabolism

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