Production and evaluation of cytotoxic effects of DT386-BR2 fusion protein as a novel anti-cancer agent.
The aim of this study was to produce a fusion protein consisting of the catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, and evaluation of its cytotoxic effects for targeted eradication of cancer cells. For this purpose, The DT386-BR2 structure was predicted using Modeller 9.14 and the best predicted model was selected based on the minimum DOPE score. A synthetic gene encoding DT386-BR2 was cloned in pET28a expression vector, expressed and purified by affinity chromatography. SDS-PAGE and Western blotting confirmed the expression of the DT386-BR2 fusion protein by revealing a band of about 47kDa after the induction of the expression. Finally, the purified protein was subjected to MTT assay for evaluation of its cyto-lethal effects on cancer and normal cell lines. Statistical analysis showed significant reduction in survival percent of HeLa and MCF-7 cancer cells in comparison to negative control (PBS), while the cytotoxic effect was not significant on the normal cells, i.e. HUVEC and HEK 293. The IC50 of DT386-BR2 for HeLa and MCF-7 was about 0.55 and 2.08μg/ml, respectively. In conclusion, the production and purification of DT386-BR2 fusion protein was successfully achieved and its cytotoxic effects on the studied cancer cell lines was established. The promising cytotoxic effects of this newly constructed fusion protein made it a suitable candidate for targeted therapy of cancer, and further in vitro and in vivo studies on this fusion protein is underway.
Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
Fatemeh
F
Shafiee
Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
Mohammad
M
Rabbani
Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran. Electronic address: jahanian@pharm.mui.ac.ir.
Ali
A
Jahanian-Najafabadi
D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004167:Diphtheria Toxin / Q000494:pharmacology*; D016503:Drug Delivery Systems / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D057809:HEK293 Cells / Q000187:drug effects; D006367:HeLa Cells / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D061986:MCF-7 Cells / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology* / Q000627:therapeutic use
D000970:Antineoplastic Agents; D004167:Diphtheria Toxin; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins
Buforin; Diphtheria toxin; Expression; Immunotoxin; Targeted therapy