Study On The Activity Of Toxin Fusion Protein DT₃₈₉-hIL13

Cancer is still a big challenge to human health, conventional treatments for cancer usually appear powerless to some tumors, and surgery has brought great suffering to patients. Therefore, an efficient and highly specific targeted therapy against tumor cells is the best strategy for tumor therapy. Toxin fusion protein can specifically kill tumor cells with a higher efficiency and fewer side effects, and shows a good prospect for anti-tumor treatment. In the previous study, we successfully found that the toxin fusion protein DT389-hIL13 specifically and efficiently killed the glioma cells which expressed IL13Rα2 in high level. The result suggested that DT389-hIL13 had a potential of being targeted drugs for tumor therapy. In order to improve the stability of DT389-hIL13 and achieve a higher biological activity, the structure of DT389-hIL13 was modified. For large-scale preparation of the fusion protein, we performed preliminary study for the downstream purification process.In our experiment, the previous recombinant expression plasmid pET-30a(+)/ DT389-hIL13 served as a template, we amplified the DT389 fragment and hIL13 fragment by PCR, and linked the two gene fragments by a new linker peptide GGGGS, which may increase the refolding efficiency by promoting the two structural domain to refold independently. At last, a new recombinant plasmid pET-30a (+)/modified DT389-hIL13 was constructed. The recombinant expression plasmid was transformed into E.coli BL21 (DE3) strain and the fusion protein was sucessfully expressed. The fusion protein was refolded and purified by Size-exclusion chromatography and ion exchange chromatography. Then we performed the proliferation inhibitory assay with U251 glioma cell to determine the biological activity of refolded fusion protein. In in vitro experiments, the refolded fusion protein was diluted to 5 concentration gradient (1×10-6M,1×10-7M,1×10-8M,1×10-9M,1×10-10M,), then the half maximal inhibitory concentrations (IC50) of the fusion protein to U251 cells were measured at three time points (24,48,72 hours). On-column refolding with size-exclusion chromatography and ion exchange chromatography was used to obtain refolded fusion proteins, we also studied the influences of different refolding and purification methods to the biological activity of fusion protein and the protein yield.We successfully constructed the pET-30a(+)/modified-DT389-hIL13 plasmid. After prokaryotic expression and purification of DT389-hIL13和modified-DT389-hIL13, we compared the purity, recovery and activity of the two fusion proteins. The purity of the fusion protein DT389-hIL13 and modified-DT389-hIL13 are respectively 89% and 85% after refolding and preliminary purification. The recoveries are respectively 45%and 40.3%. The purity of the fusion protein DT389-hIL13 and modified-DT389-hIL13 are respectively 98% and 94% after futher purification. The recoveries are respectively 37.5% and 33.3%. The IC50 to U251 cells are respectively 1.0251×10-8M and 5.1591×10-8M. Comparing the activity of DT389-hIL13 and modified-DT389-hIL13, there is not significant difference. This may be due to the unsatisfactory auxiliary role of the linker GGGGS. We also studied the influences of different refolding and purification methods to the biological activity of fusion protein and the protein yield. It was confirmed that the adsorption effect is weak between the exchanger DEAE and protein, which can reduce the loss of the purified protein. The preliminary study showed that there was no significant difference between Size-exclusion chromatography and ion exchange chromatography about protein renaturation.