Green, C.A., Kamble, N.S., Court, E.K. et al. (6 more authors) (2019) Engineering the flagellar type III secretion system: improving capacity for secretion of recombinant protein. Microbial Cell Factories, 18. 10. ISSN 1475-2859
Abstract
Background: Many valuable biopharmaceutical and biotechnological proteins have been produced in Escherichia coli, however these proteins are almost exclusively localised in the cytoplasm or periplasm. This presents challenges for purification, i.e. the removal of contaminating cellular constituents. One solution is secretion directly into the surrounding media, which we achieved via the 'hijack' of the flagellar type III secretion system (FT3SS). Ordinarily flagellar subunits are exported through the centre of the growing flagellum, before assembly at the tip. However, we exploit the fact that in the absence of certain flagellar components (e.g. cap proteins), monomeric flagellar proteins are secreted into the supernatant. Results: We report the creation and iterative improvement of an E. coli strain, by means of a modified FT3SS and a modular plasmid system, for secretion of exemplar proteins. We show that removal of the flagellin and HAP proteins (FliC and FlgKL) resulted in an optimal prototype. We next developed a high-throughput enzymatic secretion assay based on cutinase. This indicated that removal of the flagellar motor proteins, motAB (to reduce metabolic burden) and protein degradation machinery, clpX (to boost FT3SS levels intracellularly), result in high capacity secretion. We also show that a secretion construct comprising the 5'UTR and first 47 amino acidsof FliC from E. coli (but no 3'UTR) achieved the highest levels of secretion. Upon combination, we show a 24-fold improvement in secretion of a heterologous (cutinase) enzyme over the original strain. This improved strain could export a range of pharmaceutically relevant heterologous proteins [hGH, TrxA, ScFv (CH2)], achieving secreted yields of up to 0.29 mg L-1, in low cell density culture. Conclusions: We have engineered an E. coli which secretes a range of recombinant proteins, through the FT3SS, to the extracellular media. With further developments, including cell culture process strategies, we envision further improvement to the secreted titre of recombinant protein, with the potential application for protein production for biotechnological purposes.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2019 The Author(s). This 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. |
Keywords: | Recombinant protein secretion; Flagellar; Strain engineering; Secretion assay; Biotechnology; Synthetic biology |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > School of Clinical Dentistry (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 13 Mar 2019 10:00 |
Last Modified: | 26 May 2024 22:31 |
Status: | Published |
Publisher: | BMC |
Refereed: | Yes |
Identification Number: | 10.1186/s12934-019-1058-4 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:141412 |