Purification of TAT-CC-HA protein under native condition, and its transduction analysis and biological effects on BCR-ABL positive cells

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• 作者：Zhenglan Huang^a ; Maosheng Ji^a ; Zhi Peng^a ; Shifeng Huang^a ; Qing Xiao^b ; Chunli Li^a ; Jianming Zeng^a ; Miao Gao^a ; Wenli Feng^a ; ^{fengwlcqmu@sina.com"" rel=""nofollow}
• 关键词：Chronic myeloid leukemia ; Oligomerization ; TAT peptide
• 刊名：Biomedicine and Pharmacotherapy
• 出版年：2011
• 出版时间：June 2011
• 年：2011
• 卷：65
• 期：3
• 页码：183-192
• 全文大小：907 K

文摘

BCR-ABL oncoprotein is the cause of chronic myeloid leukemia. The homologous oligomerization of BCR-ABL protein mediated by BCR coiled-coil (CC) domain plays an important role in ABL kinase activation. The HIV-1 TAT peptide has been used extensively for the introduction of proteins into cells. We recombinated a TAT-CC-HA protein to interrupt the homologous oligomerization of BCR-ABL. The expression conditions for TAT-CC-HA were optimized. The TAT-CC-HA fusion protein was purified with Ni+-NTA resin. TAT-CC-HA fusion protein was added into the cultures of Ba/F3-p210, 32D-p210, K562, KU812, Ba/F3, 32D, and HL-60 cells. It was found that TAT-CC-HA could transduce into these cells. It was confirmed that TAT-CC-HA fusion protein was internalized by Ba/F3-p210, K562, and Ba/F3 cells and located in the cytoplasm observed by confocal laser scanning fluorescence microscope. The transduction of TAT-CC-HA fusion protein into K562 cells was in a dose-dependent and time-dependent manner. The result of coimmunoprecipitation assay indicated that TAT-CC-HA could interact with BCR-ABL in K562 cells. The effects of TAT-CC-HA fusion protein on cell growth and apoptosis were detected by MTT test and flow cytometry. Our findings suggested that TAT-CC-HA fusion protein could specifically inhibit the growth of BCR-ABL positive cells, and specifically induce apoptosis of BCR-ABL positive cells, while not affect the growth and apoptosis of BCR-ABL negative cells.