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Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface

Monomeric autotransporters have been extensively used for export of recombinant proteins to the cell surface of Gram-negative bacteria. A bottleneck in the biosynthesis of such constructs is the passage of the outer membrane, which is facilitated by the β-domain at the C terminus of an autotransport...

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Autores principales: Jong, Wouter S. P., Schillemans, Maaike, ten Hagen-Jongman, Corinne M., Luirink, Joen, van Ulsen, Peter
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802855/
https://www.ncbi.nlm.nih.gov/pubmed/29415042
http://dx.doi.org/10.1371/journal.pone.0191622
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author Jong, Wouter S. P.
Schillemans, Maaike
ten Hagen-Jongman, Corinne M.
Luirink, Joen
van Ulsen, Peter
author_facet Jong, Wouter S. P.
Schillemans, Maaike
ten Hagen-Jongman, Corinne M.
Luirink, Joen
van Ulsen, Peter
author_sort Jong, Wouter S. P.
collection PubMed
description Monomeric autotransporters have been extensively used for export of recombinant proteins to the cell surface of Gram-negative bacteria. A bottleneck in the biosynthesis of such constructs is the passage of the outer membrane, which is facilitated by the β-domain at the C terminus of an autotransporter in conjunction with the Bam complex in the outer membrane. We have evaluated eight β-domain constructs for their capacity to secrete fused proteins to the cell surface. These constructs derive from the monomeric autotransporters Hbp, IgA protease, Ag43 and EstA and the trimeric autotransporter Hia, which all were selected because they have been previously used for secretion of recombinant proteins. We fused three different protein domains to the eight β-domain constructs, being a Myc-tag, the Hbp passenger and a nanobody or V(HH) domain, and assessed expression, membrane insertion and surface exposure. Our results show that expression levels differed considerably between the constructs tested. The constructs that included the β-domains of Hbp and IgA protease appeared the most efficient and resulted in expression levels that were detectable on Coomassie-stained SDS-PAGE gels. The V(HH) domain appeared the most difficult fusion partner to export, probably due to its complex immunoglobulin-like structure with a tertiary structure stabilized by an intramolecular disulfide bond. Overall, the Hbp β-domain compared favorably in exporting the fused recombinant proteins, because it showed in every instance tested a good level of expression, stable membrane insertion and clear surface exposure.
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spelling pubmed-58028552018-02-23 Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface Jong, Wouter S. P. Schillemans, Maaike ten Hagen-Jongman, Corinne M. Luirink, Joen van Ulsen, Peter PLoS One Research Article Monomeric autotransporters have been extensively used for export of recombinant proteins to the cell surface of Gram-negative bacteria. A bottleneck in the biosynthesis of such constructs is the passage of the outer membrane, which is facilitated by the β-domain at the C terminus of an autotransporter in conjunction with the Bam complex in the outer membrane. We have evaluated eight β-domain constructs for their capacity to secrete fused proteins to the cell surface. These constructs derive from the monomeric autotransporters Hbp, IgA protease, Ag43 and EstA and the trimeric autotransporter Hia, which all were selected because they have been previously used for secretion of recombinant proteins. We fused three different protein domains to the eight β-domain constructs, being a Myc-tag, the Hbp passenger and a nanobody or V(HH) domain, and assessed expression, membrane insertion and surface exposure. Our results show that expression levels differed considerably between the constructs tested. The constructs that included the β-domains of Hbp and IgA protease appeared the most efficient and resulted in expression levels that were detectable on Coomassie-stained SDS-PAGE gels. The V(HH) domain appeared the most difficult fusion partner to export, probably due to its complex immunoglobulin-like structure with a tertiary structure stabilized by an intramolecular disulfide bond. Overall, the Hbp β-domain compared favorably in exporting the fused recombinant proteins, because it showed in every instance tested a good level of expression, stable membrane insertion and clear surface exposure. Public Library of Science 2018-02-07 /pmc/articles/PMC5802855/ /pubmed/29415042 http://dx.doi.org/10.1371/journal.pone.0191622 Text en © 2018 Jong et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Jong, Wouter S. P.
Schillemans, Maaike
ten Hagen-Jongman, Corinne M.
Luirink, Joen
van Ulsen, Peter
Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface
title Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface
title_full Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface
title_fullStr Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface
title_full_unstemmed Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface
title_short Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface
title_sort comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802855/
https://www.ncbi.nlm.nih.gov/pubmed/29415042
http://dx.doi.org/10.1371/journal.pone.0191622
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