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Mammalian cell-free protein expression promotes the functional characterization of the tripartite non-hemolytic enterotoxin from Bacillus cereus

Bacillus cereus is increasingly recognized as an opportunistic pathogen causing local and systemic infections. The causative strains typically produce three pore-forming enterotoxins. This study focusses on the tripartite non-hemolytic enterotoxin (Nhe). Until today, studies have tried to elucidate...

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Autores principales: Ramm, Franziska, Dondapati, Srujan Kumar, Thoring, Lena, Zemella, Anne, Wüstenhagen, Doreen Anja, Frentzel, Hendrik, Stech, Marlitt, Kubick, Stefan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031377/
https://www.ncbi.nlm.nih.gov/pubmed/32076011
http://dx.doi.org/10.1038/s41598-020-59634-8
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author Ramm, Franziska
Dondapati, Srujan Kumar
Thoring, Lena
Zemella, Anne
Wüstenhagen, Doreen Anja
Frentzel, Hendrik
Stech, Marlitt
Kubick, Stefan
author_facet Ramm, Franziska
Dondapati, Srujan Kumar
Thoring, Lena
Zemella, Anne
Wüstenhagen, Doreen Anja
Frentzel, Hendrik
Stech, Marlitt
Kubick, Stefan
author_sort Ramm, Franziska
collection PubMed
description Bacillus cereus is increasingly recognized as an opportunistic pathogen causing local and systemic infections. The causative strains typically produce three pore-forming enterotoxins. This study focusses on the tripartite non-hemolytic enterotoxin (Nhe). Until today, studies have tried to elucidate the structure, complex formation and cell binding mechanisms of the tripartite Nhe toxin. Here, we demonstrate the synthesis of the functional tripartite Nhe toxin using eukaryotic cell-free systems. Single subunits, combinations of two Nhe subunits as well as the complete tripartite toxin were tested. Functional activity was determined by hemolytic activity on sheep blood agar plates, planar lipid bilayer measurements as well as cell viability assessment using the MTT assay. Our results demonstrate that cell-free protein synthesis based on translationally active eukaryotic lysates is a platform technology for the fast and efficient synthesis of functionally active, multicomponent toxins.
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spelling pubmed-70313772020-02-27 Mammalian cell-free protein expression promotes the functional characterization of the tripartite non-hemolytic enterotoxin from Bacillus cereus Ramm, Franziska Dondapati, Srujan Kumar Thoring, Lena Zemella, Anne Wüstenhagen, Doreen Anja Frentzel, Hendrik Stech, Marlitt Kubick, Stefan Sci Rep Article Bacillus cereus is increasingly recognized as an opportunistic pathogen causing local and systemic infections. The causative strains typically produce three pore-forming enterotoxins. This study focusses on the tripartite non-hemolytic enterotoxin (Nhe). Until today, studies have tried to elucidate the structure, complex formation and cell binding mechanisms of the tripartite Nhe toxin. Here, we demonstrate the synthesis of the functional tripartite Nhe toxin using eukaryotic cell-free systems. Single subunits, combinations of two Nhe subunits as well as the complete tripartite toxin were tested. Functional activity was determined by hemolytic activity on sheep blood agar plates, planar lipid bilayer measurements as well as cell viability assessment using the MTT assay. Our results demonstrate that cell-free protein synthesis based on translationally active eukaryotic lysates is a platform technology for the fast and efficient synthesis of functionally active, multicomponent toxins. Nature Publishing Group UK 2020-02-19 /pmc/articles/PMC7031377/ /pubmed/32076011 http://dx.doi.org/10.1038/s41598-020-59634-8 Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Ramm, Franziska
Dondapati, Srujan Kumar
Thoring, Lena
Zemella, Anne
Wüstenhagen, Doreen Anja
Frentzel, Hendrik
Stech, Marlitt
Kubick, Stefan
Mammalian cell-free protein expression promotes the functional characterization of the tripartite non-hemolytic enterotoxin from Bacillus cereus
title Mammalian cell-free protein expression promotes the functional characterization of the tripartite non-hemolytic enterotoxin from Bacillus cereus
title_full Mammalian cell-free protein expression promotes the functional characterization of the tripartite non-hemolytic enterotoxin from Bacillus cereus
title_fullStr Mammalian cell-free protein expression promotes the functional characterization of the tripartite non-hemolytic enterotoxin from Bacillus cereus
title_full_unstemmed Mammalian cell-free protein expression promotes the functional characterization of the tripartite non-hemolytic enterotoxin from Bacillus cereus
title_short Mammalian cell-free protein expression promotes the functional characterization of the tripartite non-hemolytic enterotoxin from Bacillus cereus
title_sort mammalian cell-free protein expression promotes the functional characterization of the tripartite non-hemolytic enterotoxin from bacillus cereus
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031377/
https://www.ncbi.nlm.nih.gov/pubmed/32076011
http://dx.doi.org/10.1038/s41598-020-59634-8
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