Danielewicz, N, Dai, W, Rosato, F et al. (5 more authors) (2022) In-Depth Characterization of a Re-Engineered Cholera Toxin Manufacturing Process Using Growth-Decoupled Production in Escherichia coli. Toxins, 14 (6). 396. ISSN 2072-6651
Abstract
Non-toxic derivatives of the cholera toxin are extensively used in neuroscience, as neuronal tracers to reveal the location of cells in the central nervous system. They are, also, being developed as vaccine components and drug-delivery vehicles. Production of cholera-toxin derivatives is often non-reproducible; the quality and quantity require extensive fine-tuning to produce them in lab-scale settings. In our studies, we seek a resolution to this problem, by expanding the molecular toolbox of the Escherichia coli expression system with suitable production, purification, and offline analytics, to critically assess the quality of a probe or drug delivery, based on a non-toxic derivative of the cholera toxin. We present a re-engineered Cholera Toxin Complex (rCTC), wherein its toxic A1 domain was replaced with Maltose Binding Protein (MBP), as a model for an rCTC-based targeted-delivery vehicle. Here, we were able to improve the rCTC production by 11-fold (168 mg/L vs. 15 mg/L), in comparison to a host/vector combination that has been previously used (BL21(DE3) pTRBAB5-G1S). This 11-fold increase in the rCTC production capability was achieved by (1) substantial vector backbone modifications, (2) using Escherichia coli strains capable of growth-decoupling (V strains), (3) implementing a well-tuned fed-batch production protocol at a 1 L scale, and (4) testing the stability of the purified product. By an in-depth characterization of the production process, we revealed that secretion of rCTC across the E. coli Outer Membrane (OM) is processed by the Type II secretion-system general secretory pathway (gsp-operon) and that cholera toxin B-pentamerization is, likely, the rate-limiting step in complex formation. Upon successful manufacturing, we have validated the biological activity of rCTC, by measuring its binding affinity to its carbohydrate receptor GM1 oligosaccharide (Kd = 40 nM), or binding to Jurkat cells (93 pM) and delivering the cargo (MBP) in a retrograde fashion to the cell.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2022 by the authors. This is an open access article under the terms of the Creative Commons Attribution 4.0 International License (CC BY 4.0) (https://creativecommons.org/licenses/by/4.0/) |
Keywords: | cholera toxin; holotoxin-like chimaera; neuronal tracer; T2SS machinery; GM1 receptor; retrograde transport; GspD; fed-batch process; affinity chromatography |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Chemistry (Leeds) > Organic Chemistry (Leeds) |
Funding Information: | Funder Grant number EU - European Union 814029 |
Depositing User: | Symplectic Publications |
Date Deposited: | 30 Jun 2022 14:57 |
Last Modified: | 30 Jun 2022 14:57 |
Status: | Published |
Publisher: | MDPI |
Identification Number: | 10.3390/toxins14060396 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:188517 |