| Summary: | Frutalin is a plant lectin with beneficial immunobiological action, although the access to its active form is still restricted. Moreover, there is a knowledge gap on isoform activity and glycosylation impact on its bioactivity, and recombinant production protocols were seen as ineffective. Here, a simpler and faster production and purification protocol was developed, attaining a yield of purified frutalin 3.3-fold higher than that obtained previously. Hemagglutination assays confirmed that this frutalin isoform could not agglutinate rabbit erythrocytes, while maintaining the native tetrameric structure, as indicated by DLS analysis, and strong interaction with methyl-alpha-galactose, in fluorescence spectroscopy studies. The cytotoxicity of the recombinant frutalin isoform was shown in a broad panel of human cancer cells: colon (HCT116), melanoma (A375), triple-negative breast cancer (MDA-MB-231), and ovarian (IGROV-1). Treatment with 8.5–11.8 μM TrxFTL reduced proliferation of all cancer cells to half in 48 h. This anti-proliferative effect encompasses the p53 pathway since it was significantly reduced in p53-null colon cancer cells (HCT116 p53<sup>−/−</sup>; GI<sub>50</sub> of 25.0 ± 3.0 μM), when compared to the isogenic p53-positive cells (HCT116 p53<sup>+/+</sup>; GI<sub>50</sub> of 8.7 ± 1.8 μM; <i>p</i> < 0.002). This recombinantly produced frutalin isoform has relevant cytotoxic effect and its biological activity is not dependent on glycosylation. The developed <i>E. coli</i> production and purification protocol generates high yield of non-glycosylated frutalin isoform with potent cytotoxic activity, enabling the development of novel anticancer p53-targeting therapies.
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