Engineering and production of recombinant ricin protein with the aim of producing effective immunotoxin

Document Type : Research Paper

Authors

1 Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad

2 Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

3 Associate Professor, Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.

4 Professor, Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.

5 Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad,

6 Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, P. O. Box: 91775-1163, Iran

10.22103/jab.2024.23650.1578

Abstract

Aim
Ricin is a globular glycoprotein poison found in the endosperm of the castor plant. This protein consists of two A chains with 267 amino acids (32 kilo Daltons) and a B chain with 262 amino acids (34 kilodaltons), connected by a disulfide bond. The sensitivity of cancer cells to ricin is due to the higher expression of glycoproteins on the surface of cancer cells. The toxicity of the ricin enzyme in destroying cells caused this enzyme to be used as the most important candidate in the production of immunotoxins. However, the low ability of ricin to enter the target cell using the Golgi apparatus has caused increased cytotoxicity, which affected its use as an immunotoxin. This research was carried out with the aim of engineering ricin protein to increase the surface charge of the protein and facilitate the entry of ricin into the cell through the endosomal route and reduce the effect of cytotoxicity.
Methods
Therefore, the structural status of ricin protein with R134A, L214A, R31A, and P250A mutations was investigated using thermodynamic studies. Then, the gene sequence related to the mutant version was synthesized and expressed using the expression vector pET22b(+) in the cell wall of Escherichia coli BL21(DE3). Purification was done using a Ni-NTA column, and then the function of the protein on class A431 cancer epithelial cells was evaluated using a cytotoxicity test.
Results
The obtained results showed that the considered mutations significantly increased the surface charge of the protein. Also, comparing the analysis of Root Mean Square Deviation (RMSD), root-mean-square fluctuations (RMSF) and Radius of gyration (Rg) graphs between the natural sample and the mutant showed that the mutations have no significant effect on the main structure of the protein. Confirmation of the production and purification of recombinant ricin protein in the cell wall with a molecular weight of about 33 kilodaltons was done using SDS-PAGE gel. Cytotoxicity results showed that mutant ricin protein can cause cell death of A431 cancer cells at a concentration of 300 ng/ml.
Conclusion
In silico and In vitro results on ricin protein showed that creating targeted mutations to increase protein surface charge can improve protein performance. Therefore, mutant ricin protein (R134A, L214A, P250A, and R31A) with greater cell penetration and less toxicity for target cells has a high ability to be used as a toxin in the production of immunotoxins.

Keywords

References

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Agricultural Biotechnology Journal
Volume 16, Issue 4
January 2025
Pages 121-138

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