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. 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.
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Copyright, Publisher and Additional Information: | © 2023 The Protein Society. This is the peer reviewed version of the following article: 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. e4830. ISSN 0961-8368, which has been published in final form at https://doi.org/10.1002/pro.4830. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited. | ||||||
Keywords: | Human lactoferricin derived peptide; anticancer peptide; click chemistry; recombinant peptide production; peptide purification; targeted drug delivery; fusion proteins | ||||||
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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) | ||||||
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Depositing User: | Symplectic Publications | ||||||
Date Deposited: | 07 Nov 2023 15:44 | ||||||
Last Modified: | 07 Nov 2023 15:44 | ||||||
Status: | Published online | ||||||
Publisher: | Wiley | ||||||
Identification Number: | https://doi.org/10.1002/pro.4830 |
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