Expression of TAT-apoptin Fusion Protein and Its Anti-tumor Activity

Apoptin, a protein encoded by vp3 gene of chicken anemia virus, can specifically induce apoptosis of tumor cells but has no toxic effect on normal cells. To obtain transducible apoptin, the present study cloned synthesized double-stranded oligomeric nucleotide encoding TAT protein transduction domain and the gene encoding apoptin into the plasmid of pET-28a, thus generating a construct of pET-28a-TAT-apoptin which expressed TAT-apoptin fusion protein. The target protein was yielded in E. coli Rosetta(DE3)and purified by IDA-Ni~ 2+ affinity chromatography. Removal of urea via G 25 gel chromatography resulted in soluble denatured protein. Purified TAT-apoptin was then added to cultured human umbilical vein endothelial cells (HUVECs) and human lung adenocarcinoma Anip973 cells,meanwhile, TAT-MBP was added as negative control. Immuno-histochemistry showed that TAT-MBP entered cytoplasm and nucleus of the above two kinds of cells while TAT-apoptin entered cytoplasm 1 hour after protein transduction. TAT-apoptin migrated from cytoplasm to nucleus in Anip973 24 hours after transduction; in the contrast, there were no changes in subcelluar localization of TAT-apoptin in HUVECs and TAT-MBP in above two kinds of cells. TUNEL assay revealed that massive apoptotic cells could be found in Anip973 cells treated with TAT-apoptin, while no obvious changes occurred to HUVECs treated with TAT-MBP or TAT-apoptin and Anip973 cells treated with TAT-MBP. The results suggest that TAT-apoptin fusion protein may possess potential value in tumor therapy.