Bioinformatic design and expression validation of cholera toxin B-linker-enterotoxin A-ligand Produced by Populus euramericana (Dode) Guinier

Shahghobadi, H; Etemad, V; Esmaili, D; Shirvani, A

doi:10.22034/ijf.2025.489445.2017

Bioinformatic design and expression validation of cholera toxin B-linker-enterotoxin A-ligand Produced by Populus euramericana (Dode) Guinier

Document Type : Research Paper

Authors

H Shahghobadi ¹

V Etemad ²

D Esmaili ³

A Shirvani ²

¹ PhD. Dept. of Forestry and Forest Economics, Faculty of Natural Resources, University of Tehran, Karaj, Iran

² Associate Prof., Dept. of Forestry and Forest Economics, Faculty of Natural Resources, University of Tehran, Karaj, Iran

³ Prof., Dept. of Medical Bacteriology, Tarbiat Modares University, Tehran, Iran

10.22034/ijf.2025.489445.2017

Abstract

Introduction: Due to the increasing drug resistance, there is a critical need to identify novel anticancer agents and develop innovative therapeutic strategies. Many bacteria produce peptides known as toxins, which exhibit antitumor effects on various cancer cell lines. Despite significant advancements in cancer therapy, the limitations associated with conventional anticancer agents have directed research toward natural compounds, particularly enterotoxins. The objective of this study was to design and verify the expression of the Anticancer‑cTxB‑linker‑enTA‑Ligand fusion protein in Populus euramericana, one of the most economically significant tree species. This species is highly valued for its rapid growth, making it a viable candidate for medium- to long-term investment.
Materials and Methods: To design the target protein, the sequences of enterotoxin A and cholera toxin B were retrieved from the NCBI genomic database. A linker sequence was inserted between the two enterotoxin sequences to enhance flexibility and maintain structural integrity, and an anticancer sequence was added to the gene to boost antitumor activity. Finally, the recombinant fusion gene was synthesized, and expression and purification processes were conducted. The expression of the target recombinant protein was verified using SDS‑PAGE and Western blot.
Results: The results demonstrated that the designed fusion protein possesses a suitable structure and favorable physicochemical stability, showing high resistance under simulated aqueous conditions. Furthermore, the successful transfer of the recombinant vector was confirmed using colony PCR, and the observation of a distinct 238 bp band indicated the presence of the recombinant fusion gene. SDS‑PAGE and Western blot results clearly confirmed the successful expression of the recombinant protein, with a molecular weight of 47 kDa, in poplar leaves.
Conclusion: The fusion protein, derived from the combination of enterotoxin A, cholera toxin B, and the anticancer sequence, exhibits significant antitumor properties. Therefore, preclinical evaluations, including animal studies, are currently underway to assess the potential of this compound as a complementary or alternative strategy in cancer therapy.

Keywords

Agroinfiltration

Anticancer activity

Enterotoxin

Subjects

Plant Physiology and Genetic

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