利用类弹性蛋白可逆相变和内含肽自切割功能高效制备TmSm抗肿瘤蛋白  

Efficient Preparation of an Anti-tumor Protein TmSm by Reversible Phase Transition of ELPs and Self-cleaving Function of Intein

作　　者：石磊[1] 唐莉莉[1] 马兴元[1] 王天文[1] 马飞[1] 王平[1] 

机构地区：[1]华东理工大学生物工程学院生物反应器工程国家重点实验室,上海200237

出　　处：《中国生物工程杂志》2013年第10期89-95,共7页China Biotechnology

基　　金：国家自然科学基金(30873190);"十一五"新药创制(2009ZX09103-693);教育部中央高校基本科研业务费(WK0913002)资助项目

摘　　要：目标蛋白TmSm是研发出的一种能够较强抑制肿瘤细胞生长和促进其凋亡的基因工程抗肿瘤蛋白,其由介导目的蛋白进入细胞的转导肽HIV-TAT突变体(Tm)和Survivin突变体(Sm)融合而成。原先的TmSm制备工艺以包涵体表达、变性和复性为主,其过程不仅复杂、收率低成本高,而且所得蛋白活性较低。通过将TmSm基因与编码类弹性蛋白(elastin-like polypeptides,ELPs)和内含肽(intein)的标签序列(EI)进行融合,利用ELPs的可逆相变特性和intein的自切割功能,经简单的温度及pH调控,获得了纯度为99.00%的TmSm蛋白。将纯化后的蛋白作用于肺癌细胞A549,24 h后MTT结果显示,63.21μg/ml的目的蛋白对A549的抑制率为57.83%;流式细胞仪检测结果表明,当TmSm浓度为40μg/ml时,24 h后细胞凋亡率为41.72%。因此,这种新型的以类弹性蛋白和内含肽介导的表达纯化技术不仅成本低廉,而且对抗肿瘤蛋白药物的质量提高具有重要应用价值。The target protein TmSm developed is a genetically engineered anti-tumor protein that can inhibit the growth and promote the apoptosis of tumor cells. TmSm is generated by fusion of the cell-penetrating peptide HIV-TAT （Tm） and Survivin mutant （Sm）. The previous preparation of TmSm was based on traditional processes including expression, denaturation and refolding of inclusion body. These processes are not only complicated and high-cost, but also resulted in the low activity of recombinant proteins. Therefove TmSm gene was fused with hybrid tag sequence encoding elastin-like polypeptides （ELPs） and intein. Preparation of protein TmSm was mainly achieved by using reversible phase transition of ELPs and self-cleaving ability of intein. At last, the protein purity of 99.00% by the simple operation of temperature and pH was obtained. After 24 h treatment of lung cancer A549 cells with purified TmSm, MTT assays showed that the inhibitory rate for A549 was 57.83% with the TmSm concentration of 63.21 μg/ml; flow cytometry analysis indicated that the inhibitory rate for A549 cell was 41.72% with the TmSm concentration of 40 μg/ml. Therefore, this novel protein preparation technique, i.e. ELPs and intein-mediated purification system, is not only low-cost, but also has significant application value in improving the quality of the anti-tumor protein drug.

关 键 词：类弹性蛋白 内含肽 重组蛋白TmSm 高效制备 肺癌细胞A549 

分 类 号：Q819[生物学—生物工程]