Determining Induction Conditions for Expression of Truncated Diphtheria Toxin and Pseudomonas Exotoxin A in E. coli BL21

Targeted cancer therapies have played a great role in the treatment of malignant tumors, in the recent years. Among these therapies, targeted toxin therapies, immunotoxins, has improved the patient’s survival rate by minimizing the adverse effect on normal tissues, whereas delivering a high dose of tumoricidal agent for eradicating the cancer tissue. Immunologic proteins such as antibodies are conjugated to plant toxins or bacterial toxins such as Diphtheria toxin (DT) and Pseudomonas exotoxin A (PEA). In this case optimizing and expressing the Diphtheria toxin and Pseudomonas exotoxin A which their binding domains are eliminated plays a crucial role in producing the desired immunotoxin
We expressed the truncated DT and PE toxin in a genetically modified E.coli strain BL21 (DE3). For this reason we eliminated the binding domain sequence of these toxins and expressed these proteins in an expression vector pET28a with the kanamycin resistant gene for selection. The optimization of the Diphtheria toxin and Pseudomonas exotoxin A expression were due to different IPTG concentration, induction and sonication time
We observed that the optimal protein expression was gained in 4 hours and at 25°C with 0.4 mM IPTG concentration for DT and 4 hours and at 25°C with 0.5 mM IPTG concentration for PEA.
Our study also showed lower IPTG concentrations could result in higher protein expression. By optimizing this procedure, we facilitate the protein production which could lead to accelerate the drug development.