Immunotoxin is a new strategy for protein therapy of cancer. This engineered protein contains two parts, the immune part which is an antibody or cytokine, directed against the cancer cell receptor, and the toxin part consisting of a plant or bacterial toxin leading to apoptosis by protein synthesis inhibition. The knowledge of cell-surface receptor overexpression in cancer cells can help scientists to construct new anti-cancer agents. The granulocyte colony stimulating factor (G-CSF) receptor is expressed on the cell surface of some blood cancers such as acute myeloid leukemia (AML). Therefore, this receptor can be used as an immunotoxin for treatment of some cancers. The aim of this work was to design and produce DT-GCSF immunotoxin using truncated DT fused to G-CSF. For fusion protein construction, DT389 and G-CSF fragments, were amplified by PCR using specific primers. A flexible linker SerGly4SerMet (SG4SM) was used to fuse the PCR products by SOEing PCR procedure to achieve an appropriate fusion protein, and the fused fragment was subcloned into pET21b. The new construction (pET-DT389GCSF) was transformed into E. coli strain BL21 (DE3) and the expression of the construction was confirmed by SDS-PAGE and Western blotting techniques. The data demonstrated the expression and purity rates of DT389GCSF about 25% and 90%, respectively. This chimeric protein construction can be used as a new anti-AML drug, but its in vitro and in vivo biological activity should be analyzed.
Keywords: Diphtheria toxin; Expression; Granulocyte colony-stimulating factor; Immunotoxin; Purification.