Pasupuleti, R., Riedl, S., Saltor Núñez, L. et al. (7 more authors) (2023) Lectin-anticancer peptide fusion demonstrates significant cancer-cell-selective cytotoxic effect and inspires the production of ‘clickable’ anticancer peptide in E. coli. Protein Science, 32 (12). e4830. ISSN 0961-8368
Abstract
Targeted killing of tumor cells while protecting healthy cells is the pressing priority in cancer treatment. Lectins that target a specific glycan marker abundant on cancer cells can be valuable new tools for selective cancer cell killing. The lectin shiga-like toxin 1 B subunit (Stx1B) is an example that specifically binds globotriaosylceramide (CD77 or Gb3), which is overexpressed in certain cancers. In this study, a human lactoferricin-derived synthetic retro di-peptide R-DIM-P-LF11-215 with antitumor efficacy was fused to the lectin Stx1B to selectively target and kill Gb3+ cancer cells. We produced lectin-peptide fusion proteins in E. coli, isolated them by Gb3-affinity chromatography and assessed their ability to selectively kill Gb3+ cancer cells in a Calcein AM assay. Furthermore, to expand the applications of R-DIM-P-LF11-215 in developing therapeutic bioconjugates, we labelled R-DIM-P-LF11-215 with the unique reactive non-canonical amino acid Nε-((2-azidoethoxy)carbonyl)-L-lysine (AzK) at a selected position by amber stop codon suppression. The R-DIM-P-LF11-215 20AzK and the unlabeled R-DIM-P-LF11-215 parent peptide were produced as GST-fusion proteins for soluble expression in E. coli for the first time. We purified both variants by size-exclusion chromatography and analyzed their peptide masses. Finally, a cyanin 3 fluorophore was covalently conjugated to R-DIM-P-LF11-215 20AzK by strain-promoted alkyne-azide cycloaddition. Our results showed that the recombinant lectin-peptide fusion R-DIM-P-LF11-215-Stx1B killed >99% Gb3+ HeLa cells while Gb3-negative cells were unaffected. The peptides R-DIM-P-LF11-215 and R-DIM-P-LF11-215 20AzK were produced recombinantly in E. coli in satisfactory amounts and were tested functional by cytotoxicity and cell-binding assays, respectively.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2023 The Authors. This is an open access article under the terms of the he Creative Commons Attribution-NonCommercial-NoDerivs License (CC-BY-NC-ND 4.0). |
Keywords: | Human lactoferricin derived peptide; anticancer peptide; click chemistry; recombinant peptide production; peptide purification; targeted drug delivery; fusion proteins |
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 EU - European Union 746421 |
Depositing User: | Symplectic Publications |
Date Deposited: | 07 Nov 2023 15:44 |
Last Modified: | 09 Dec 2024 15:56 |
Status: | Published |
Publisher: | Wiley |
Identification Number: | 10.1002/pro.4830 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:204966 |