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Expression, purification and characterization of recombinant toxins consisting of truncated gastrin 17 and
Xiao-Li Feng, Xi-Lin Liu, Shi-Ying Lu
1Key Laboratory of Zoonosis Research, Ministry of Education / Institute of Zoonosis / College of Veterinary Medicine, Jilin University, Xi An Da Lu 5333, Changchun 130062, China. zsliu1959@163.com.
Abstract:
Gastric cancer is a major cause of mortality and morbidity around world. However the effectiveness of the current approaches to the diagnosis and treatment of gastric cancer is limited. Recombinant targeted toxins may represent a novel direction of cancer therapy. In this study, we aimed to explore whether recombinant toxins fused with the truncated forms of G17 could target to kill cancer cells by recognizing CCK2R. Four recombinant Pseudomonas toxins PE38 fused with the forward or reverse truncated forms of G17 (G14 and G13) were successfully constructed, expressed, and purified. Their characteristics were further analyzed by SDS-PAGE, western blot and indirect immunofluorescence assay. The cytotoxicity assay demonstrated that only reversely fused recombinant toxins rG14PE38 and rG13PE38 exhibited certain toxicity on several cancer cell lines, and a competition assay indicated that the binding of the reverse gastrin-endotoxin to CCK2R (+) cells may be mediated by interaction between gastrin/gastrin-like and CCK2R.
Insights
Novel recombinant toxins targeting gastric cancer cells show promise. Researchers fused Pseudomonas exotoxin A (PE38) with gastrin fragments, finding reverse fusions effectively killed cancer cells by targeting CCK2R.
Area of Science:
- Oncology
- Molecular Biology
- Biotechnology
Background:
- Gastric cancer presents a significant global health challenge with limited therapeutic options.
- Recombinant targeted toxins offer a potential novel strategy for cancer treatment.
Purpose of the Study:
- To investigate the efficacy of recombinant toxins, specifically Pseudomonas exotoxin A (PE38) fused with truncated gastrin-17 (G17), in targeting and eliminating cancer cells.
- To determine if these constructs recognize and bind to cholecystokinin B receptors (CCK2R) expressed on cancer cells.
Main Methods:
- Construction, expression, and purification of four recombinant PE38 toxins fused with forward or reverse truncated G17 (G14 and G13).
- Characterization of recombinant toxins using SDS-PAGE, western blot, and indirect immunofluorescence assays.
- Assessment of cytotoxicity against cancer cell lines and competition assays to evaluate CCK2R binding specificity.
Main Results:
- Successfully generated and characterized four distinct recombinant gastrin-PE38 fusion toxins.
- Recombinant toxins with reverse fusion of truncated gastrin (rG14PE38 and rG13PE38) demonstrated significant cytotoxicity against tested cancer cell lines.
- Competition assays suggested that the binding of these reverse fusion toxins to CCK2R-positive cells is mediated by gastrin/gastrin-like interactions with CCK2R.
Conclusions:
- Recombinant toxins engineered with reverse-fused gastrin fragments show targeted toxicity towards gastric cancer cells.
- The CCK2R receptor is a viable target for these novel gastrin-based targeted toxins in gastric cancer therapy.
- Further development of these targeted toxins could lead to improved gastric cancer treatment strategies.