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生物工程学报 2006
Preparation of Recombinant HIV-TATm-survivin (T34A) Protein and its Pro-apoptosis Activity to Cancer Cells in vitro
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Abstract:
As a novel member of the IAP (Inhibitor of apoptosis protein) family, survivin was observed to be expressed in most human cancerous cells. Fusion protein TATm-survivin (T34A) has drawn considerable attention because it is a potential anti-tumor protein that can be transduced into cancer cell with the help of HIV-TAT domain. In this study, the cDNA encoding survivin was cloned by RT-PCR from human breast cancer cell lines B-Cap-37. An expression vector of pRSET-B-HIV-tatm-survivin (T34A) was constructed by PCR after survivin (T34A) was mutated by site-directed mutagenesis. Subsequently, the resultant plasmid was transformed into E. coli BL21 (DE3). Recombinant HIV-TATm-Survivin (T34A) protein was expressed efficiently with 0.5mM IPTG as inducer, reaching a yield of 650mg/liter (as inclusion body) in fermentation culture. The inclusion bodies were solubilized, refolded and purified to a purity of 96% by ion exchange chromatograghy and size-exclusion chromatography. Remarkable effects of the purified recombinant HIV-TATm-Survivin (T34A) on the morphology of cell line SW1990 and B-Cap-37 were observed after being administrated for 4h. MTT assay showed recombinant HIV-TATm-survivin (T34A) protein could inhibit significantly cell proliferation of SW1990 and B-Cap-37 and SSMC-7721 in vitro. Apoptosis rate and cell circle of SW1990 and B-Cap-37 that had been treated with target protein (final concentration 30 microg/mL) were detected with flow cytometry. Results revealed that more than 65% cancer cells were arrested at G1 phase. The study suggested that TATm-survivin (T34A) protein was a hopeful protein drug in the treatment of cancers by facilitating apoptosis of cancer cells. Key words recombinant HIV-TATm-Survivin (T34A), expression and purification, pro-apoptosis bioactivity, SW1990 and B-Cap-37 cancer cell lines
