Skip to main content

Lund University Publications

LUND UNIVERSITY LIBRARIES

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

Lindh, Tova LU ; Collin, Mattias LU orcid ; Lood, Rolf LU and Carlquist, Magnus LU (2023) In Methods in molecular biology (Clifton, N.J.) p.131-146
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... (More)

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.

(Less)
Please use this url to cite or link to this publication:
author
; ; and
organization
publishing date
type
Chapter in Book/Report/Conference proceeding
publication status
published
subject
keywords
Saccharomyces cerevisiae/genetics, Bacterial Proteins/metabolism, Recombinant Fusion Proteins/genetics, Fluorescence, Peptide Hydrolases/metabolism
host publication
Bacterial pathogenesis : Methods and protocols - Methods and protocols
series title
Methods in molecular biology (Clifton, N.J.)
editor
Nordenfelt, Pontus and Collin, Mattias
edition
2nd
pages
131 - 146
publisher
Humana Press
external identifiers
  • pmid:37258965
  • scopus:85160781455
ISSN
1940-6029
ISBN
978-1-0716-3243-7
DOI
10.1007/978-1-0716-3243-7_9
language
English
LU publication?
yes
id
524a6104-c032-48a9-a4b1-bcaac8268395
date added to LUP
2023-06-07 09:35:49
date last changed
2024-06-15 03:48:21
@inbook{524a6104-c032-48a9-a4b1-bcaac8268395,
  abstract     = {{<p>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.</p>}},
  author       = {{Lindh, Tova and Collin, Mattias and Lood, Rolf and Carlquist, Magnus}},
  booktitle    = {{Bacterial pathogenesis : Methods and protocols}},
  editor       = {{Nordenfelt, Pontus and Collin, Mattias}},
  isbn         = {{978-1-0716-3243-7}},
  issn         = {{1940-6029}},
  keywords     = {{Saccharomyces cerevisiae/genetics; Bacterial Proteins/metabolism; Recombinant Fusion Proteins/genetics; Fluorescence; Peptide Hydrolases/metabolism}},
  language     = {{eng}},
  pages        = {{131--146}},
  publisher    = {{Humana Press}},
  series       = {{Methods in molecular biology (Clifton, N.J.)}},
  title        = {{Expression of the Bacterial Enzyme IdeS Using a GFP Fusion in the Yeast Saccharomyces cerevisiae}},
  url          = {{http://dx.doi.org/10.1007/978-1-0716-3243-7_9}},
  doi          = {{10.1007/978-1-0716-3243-7_9}},
  year         = {{2023}},
}