A new strategy for the high-yield production of recombinant Clostridium septicum alpha-protoxin: Periplasmic expression in Escherichia coli C43 followed by extracellular release via osmotic shock

Esmaelizad, Majid; Seyedi, Sahar; Noofeli, Mojtaba; Angaji, Abdolhamid; Nazari, Ali; Aetemady, Afshar; Khoshnood, Siamak; Samianifard, Maedeh

doi:10.66224/ari.2026.372777.4118

A new strategy for the high-yield production of recombinant Clostridium septicum alpha-protoxin: Periplasmic expression in Escherichia coli C43 followed by extracellular release via osmotic shock

Articles in Press

Document Type : Original Articles

Authors

¹ Research and Development Department, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Alborz, Iran.

² Department of Biology, Faculty of Biological sciences, Kharazmi University, Tehran, Iran

³ Research and Development Department, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Alborz, Iran

10.66224/ari.2026.372777.4118

Abstract

Introduction: Given the challenges associated with producing native toxin from Clostridium bacteria, including biosafety risks, impurity and low yield, recombinant expression in hosts such as E. coli has emerged as a safe, pure and cost-effective approach. Materials & Methods: The codon-optimized sequence of the alpha protoxin of Clostridium septicum was synthesized and cloned into the pET22b+ vector for expression in E. coli. The recombinant plasmid was transformed into the E. coli strain C43 via heat shock and induced with 0.2% lactose in 5 liter 2YT media at 22°C. Using the pelB leader signal, the protoxin was expressed in the periplasmic space. Osmotic shock was employed to release the protein extracellularly. The protoxin was purified from the osmotic shock buffer containing the recombinant toxin using nickel resin affinity chromatography. Results: The yield of purification estimated 10.4mg per one liter of bacterial culture. After dialysis, protein concentration was performed using the Bradford assay. The recombinant protoxin was confirmed by Western blotting and specific NIBSC antibody by indirect ELISA. Furthermore, the produced recombinant toxin successfully detected antibodies generated in rabbits immunized with the quadrivalent clostridial vaccine of Razi institute using indirect ELISA assay. Conclusion: This recombinant toxin might be used for the development of recombinant vaccine or as a standard antigen for quality control of the traditional vaccine.

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