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...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2017
|
Materias: | |
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 |
_version_ | 1783288105012297728 |
---|---|
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. |
format | Online Article Text |
id | pubmed-5740907 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT chengcheng anovelffufusionsystemforsecretoryexpressionofheterologousproteinsinescherichiacoli AT wushanshan anovelffufusionsystemforsecretoryexpressionofheterologousproteinsinescherichiacoli AT cuilupeng anovelffufusionsystemforsecretoryexpressionofheterologousproteinsinescherichiacoli AT wuyulu anovelffufusionsystemforsecretoryexpressionofheterologousproteinsinescherichiacoli AT jiangtianyue anovelffufusionsystemforsecretoryexpressionofheterologousproteinsinescherichiacoli AT hebingfang anovelffufusionsystemforsecretoryexpressionofheterologousproteinsinescherichiacoli AT chengcheng novelffufusionsystemforsecretoryexpressionofheterologousproteinsinescherichiacoli AT wushanshan novelffufusionsystemforsecretoryexpressionofheterologousproteinsinescherichiacoli AT cuilupeng novelffufusionsystemforsecretoryexpressionofheterologousproteinsinescherichiacoli AT wuyulu novelffufusionsystemforsecretoryexpressionofheterologousproteinsinescherichiacoli AT jiangtianyue novelffufusionsystemforsecretoryexpressionofheterologousproteinsinescherichiacoli AT hebingfang novelffufusionsystemforsecretoryexpressionofheterologousproteinsinescherichiacoli |