Design and Expression of Recombinant cell Penetrating Protein Based on Tat and pertussis-like Toxin A and Evaluation of its Effects on the lung cancer

Abstract

Nowadays, design of cytotoxic agents based on microbial toxins is attracted for researchers. Pertussis-like toxin subunit A (PltA) of typhoid toxin is ADP-ribosyl transferase and had the cytotoxicity and cell arrest property in G2/M phase of human cancer cell line. To translocate and its increase to the cells, PltA requires the cell penetrating part. Here, the catalytic PltA (named Typh) was attached to Tat peptide as a cell penetrating agent and expressed as a new recombinant fusion protein (named Tat-Typh) in E. coli BL21. After that, recombinant Tat-Typh was purified using Ni + chromatography column and confirmed by western blotting. Finally, its cytotoxicity effects and cell penetration activity were evaluated by, MTT assay, Annexin-V/PI staining and western blotting methods, respectively. Our results showed that Tat-Typh had the significant cytotoxic effect at 25, 50, 150 and 200 µg/mL concentrations (P < 0.05). In addition, cell treating with 50 µg/mL Tat-Typh was resulted in to increase the percentage of necrotic cells compared to control groups (P < 0.05). Moreover, binding of Tat fragment to Typh protein caused to increase the speed of entry of Tat-Typh to cells compared to Typh alone. In conclusion, it is observed that Tat-Typh protein is able to increase the cell penetration properties of catalytic Pertussis-like toxin subunit A and has the cytotoxic effects on lung cancer cell line.