Objective Immunotoxins are one of the most promising ways in therapeutic fields, specially cancer therapy which have a unique toxin-antibody structure, and kill the cancer cell by passing through the cell membrane and entering the target cell. The specificity of immunotoxins for target cell selection is dependent on the selected antibody part and the cytotoxic effect to induce apoptosis in target cells is due to the toxin moiety. The aim of this study was to engineer and design bovine pancreatic enzyme (RNase A) for to escape from ribonuclease inhibitor (RI) as a toxin segment in designing of immunotoxins used in therapeutic fields. Materials and Methods After bioinformatics investigation using PyMol software, engineering and substitution of target amino acid in wild gene coding RNase A sequence (K91A) were performed. The wild recombinant proteins and engineered in E.coli (BL21(D3) strain) were expressed and refolded, and then approved using SDS-PAGE. The purification of produced proteins was conducted using an immobilized-metal affinity chromatography for His-tagged proteins. Results The results showed that the protein concentrations were 3 and 2.2 g/L for wild RNase A and engineered variant. The hydrolysis activity measurement of the produced enzymes, in presence of different concentrations of RI, indicated that engineered variant had lower sensitivity and higher catalytic activity in comparison with the wild enzyme. The results obtained from enzyme activity of engineered enzyme compared to the wild enzyme, in presence or absence of RI, demonstrated that the interaction effect between RNase A and RI can be disrupted by manipulation of amino acids involved in this interaction. Conclusion In respect to the current results, it was concluded that bovine pancreatic ribonuclease (RNase A) enzyme can be used as a promising drug in engineering and production of immunotoxins.

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