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

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 recombinant mature epsilon toxin to evaluate cell death impact on the kidney cell line. For this purpose, the sequence of mature epsilon toxin (46-328 aa) in pET28a was cloned and expressed in 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 concentrations 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. Induction by 1 mM IPTG for 4 h at 37°C was an optimized condition for expressing mature epsilon toxin in 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 ( ≤0.05). The considerable increase of necrotic cell percentage was shown after exposing to 100, 150, and 200 μg/mL of mature epsilon toxin ( ≤0.05). The recombinant mature epsilon toxin had cytotoxic effects and could induce necrosis.