A novel Ffu fusion system for secretory expression of heterologous proteins in Escherichia coli

BACKGROUND: The high level of excretion and rapid folding ability of β-fructofuranosidase (β-FFase) in Escherichia coli has suggested that β-FFase from Arthrobacter arilaitensis NJEM01 can be developed as a fusion partner. METHODS: Based on the modified Wilkinson and Harrison algorithm and the preli...

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Autores principales: Cheng, Cheng, Wu, Shanshan, Cui, Lupeng, Wu, Yulu, Jiang, Tianyue, He, Bingfang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5740907/
https://www.ncbi.nlm.nih.gov/pubmed/29268791
http://dx.doi.org/10.1186/s12934-017-0845-z
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author Cheng, Cheng
Wu, Shanshan
Cui, Lupeng
Wu, Yulu
Jiang, Tianyue
He, Bingfang
author_facet Cheng, Cheng
Wu, Shanshan
Cui, Lupeng
Wu, Yulu
Jiang, Tianyue
He, Bingfang
author_sort Cheng, Cheng
collection PubMed
description BACKGROUND: The high level of excretion and rapid folding ability of β-fructofuranosidase (β-FFase) in Escherichia coli has suggested that β-FFase from Arthrobacter arilaitensis NJEM01 can be developed as a fusion partner. METHODS: Based on the modified Wilkinson and Harrison algorithm and the preliminary verification of the solubility-enhancing ability of β-FFase truncations, three β-FFase truncations (i.e., Ffu209, Ffu217, and Ffu312) with a native signal peptide were selected as novel Ffu fusion tags. Four difficult-to-express protein models; i.e., CARDS TX, VEGFR-2, RVs and Omp85 were used in the assessment of Ffu fusion tags. RESULTS: The expression levels and solubility of each protein were markedly enhanced by the Ffu fusion system. Each protein had a favorable Ffu tag. The Ffu fusion tags performed preferably when compared with the well-known fusion tags MBP and NusA. Strikingly, it was confirmed that Ffu fusion proteins were secreted into the periplasm by the periplasmic analysis and N-amino acid sequence analysis. Further, efficient excretion of HV3 with defined anti-thrombin activity was obtained when it was fused with the Ffu312 tag. Moreover, HV3 remained soluble and demonstrated notable anti-thrombin activity after the removal of the Ffu312 tag by enterokinase. CONCLUSIONS: Observations from this work not only complements fusion technologies, but also develops a novel and effective secretory system to solve key issues that include inclusion bodies and degradation when expressing heterologous proteins in E. coli, especially for proteins that require disulfide bond formation, eukaryotic-secreted proteins, and membrane-associated proteins. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-017-0845-z) contains supplementary material, which is available to authorized users.
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spelling pubmed-57409072018-01-03 A novel Ffu fusion system for secretory expression of heterologous proteins in Escherichia coli Cheng, Cheng Wu, Shanshan Cui, Lupeng Wu, Yulu Jiang, Tianyue He, Bingfang Microb Cell Fact Research BACKGROUND: The high level of excretion and rapid folding ability of β-fructofuranosidase (β-FFase) in Escherichia coli has suggested that β-FFase from Arthrobacter arilaitensis NJEM01 can be developed as a fusion partner. METHODS: Based on the modified Wilkinson and Harrison algorithm and the preliminary verification of the solubility-enhancing ability of β-FFase truncations, three β-FFase truncations (i.e., Ffu209, Ffu217, and Ffu312) with a native signal peptide were selected as novel Ffu fusion tags. Four difficult-to-express protein models; i.e., CARDS TX, VEGFR-2, RVs and Omp85 were used in the assessment of Ffu fusion tags. RESULTS: The expression levels and solubility of each protein were markedly enhanced by the Ffu fusion system. Each protein had a favorable Ffu tag. The Ffu fusion tags performed preferably when compared with the well-known fusion tags MBP and NusA. Strikingly, it was confirmed that Ffu fusion proteins were secreted into the periplasm by the periplasmic analysis and N-amino acid sequence analysis. Further, efficient excretion of HV3 with defined anti-thrombin activity was obtained when it was fused with the Ffu312 tag. Moreover, HV3 remained soluble and demonstrated notable anti-thrombin activity after the removal of the Ffu312 tag by enterokinase. CONCLUSIONS: Observations from this work not only complements fusion technologies, but also develops a novel and effective secretory system to solve key issues that include inclusion bodies and degradation when expressing heterologous proteins in E. coli, especially for proteins that require disulfide bond formation, eukaryotic-secreted proteins, and membrane-associated proteins. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-017-0845-z) contains supplementary material, which is available to authorized users. BioMed Central 2017-12-21 /pmc/articles/PMC5740907/ /pubmed/29268791 http://dx.doi.org/10.1186/s12934-017-0845-z Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided 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 Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Cheng, Cheng
Wu, Shanshan
Cui, Lupeng
Wu, Yulu
Jiang, Tianyue
He, Bingfang
A novel Ffu fusion system for secretory expression of heterologous proteins in Escherichia coli
title A novel Ffu fusion system for secretory expression of heterologous proteins in Escherichia coli
title_full A novel Ffu fusion system for secretory expression of heterologous proteins in Escherichia coli
title_fullStr A novel Ffu fusion system for secretory expression of heterologous proteins in Escherichia coli
title_full_unstemmed A novel Ffu fusion system for secretory expression of heterologous proteins in Escherichia coli
title_short A novel Ffu fusion system for secretory expression of heterologous proteins in Escherichia coli
title_sort novel ffu fusion system for secretory expression of heterologous proteins in escherichia coli
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5740907/
https://www.ncbi.nlm.nih.gov/pubmed/29268791
http://dx.doi.org/10.1186/s12934-017-0845-z
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