Cell death analysis of recombinant mature epsilon toxin on the kidney cell line

Roza Chehreara; Shohreh  Zare Karizi; Hamideh Mahmoodzadeh Hosseini; Seyed Ali Mirhosseini; Mohammad Shafiei; Jafar Amani; Rouhollah Kazemi

doi:10.18502/ijm.v13i6.8088

Roza Chehreara Department of Genetics, Faculty of Basic Sciences, Islamic Azad University, Research Branch, Tehran, Iran
Shohreh Zare Karizi Department of Genetics and Biotechnology, School of Biological Science, Varamin-Pishva, Branch Islamic Azad University, Varamin, Iran
Hamideh Mahmoodzadeh Hosseini Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
Seyed Ali Mirhosseini Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
Mohammad Shafiei Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
Jafar Amani Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
Rouhollah Kazemi Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

DOI: https://doi.org/10.18502/ijm.v13i6.8088

Keywords: Cell death; Clostridium perfringens; Epsilon toxin; Necrosis; Recombinant expression

Abstract

Background and Objectives: Epsilon toxin is the third hazardous bacterial toxin causing ABS enterotoxaemia in domestic animal. In addition, epsilon toxin is known as a biological warfare agent. The aim of this study was to produce the recombi- nant mature epsilon toxin to evaluate cell death impact on the kidney cell line.

Materials and Methods: For this purpose, the sequence of mature epsilon toxin (46-328 aa) in pET28a was cloned and expressed in Escherichia coli BL21 (DE3) and purified by nickel-nitrilotriacetic acid (Ni-NTA) column and confirmed by western blot analysis using HRP conjugated anti-His antibody. Then, to assess the anti-proliferative effects of different con- centrations of recombinant epsilon toxin, the MTT assay was done on the HEK293 cell line. The annexin V/PI staining was done to investigate the apoptotic and necrotic cell populations after exposure to epsilon toxin.

Results: Induction by 1 mM IPTG for 4 h at 37°C was an optimized condition for expressing mature epsilon toxin in E. coli strain BL21 (DE3). Electrophoresis on SDS-PAGE 12% gel showed the desired band approximately at 38 KDa. Our results showed that recombinant epsilon toxin is mainly expressed as an inclusion body. Furthermore, 100, 150, and 200 µg/mL of mature epsilon toxin are significantly reduced the cell viability (P≤0.05). The considerable increase of necrotic cell percent- age was shown after exposing to 100, 150, and 200 µg/mL of mature epsilon toxin (P≤0.05).

Conclusion: The recombinant mature epsilon toxin had cytotoxic effects and could induce necrosis.