细胞穿膜肽TAT与小鼠干细胞转录因子Oct4融合表达及纯化

Expression and Purification of Cell Penetrating Peptide TAT and Mouse Stem Cell Transcriptional Factor Oct4 Recombinant Protein

DOI：10.3969/j.issn.1673-1689.2013.09.011

中文关键词: 细胞穿膜肽 干细胞转录因子Oct4 包涵体 纯化

英文关键词: TAT Oct4 inclusion body purification

基金项目:国家自然科学基金项目(81273437);中国科学院战略性先导科技专项(XDA01040200)

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中文摘要:

将TAT-Oct4目的基因插入pET28a表达载体,构建重组表达质粒pET28a-TAT-Oct4,形成大肠杆菌重组表达体系。融合蛋白TAT-Oct4在大肠杆菌中主要以包涵体形式表达,表达产物经纯化后,纯度可达90%以上。通过尿素梯度透析复性,TAT-Oct4平均复性收率为8.8%。细胞免疫荧光技术分析表明,TAT-Oct4融合蛋白可穿透近100%细胞的细胞膜,其进膜后主要分布于细胞核内。建立了具有活性的TAT-Oct4融合蛋白生物制备方法,为细胞重编程提供了一种有效的研究材料,并可为其他用于细胞重编程的干细胞转录因子设计提供实用的方法学。

英文摘要:

In this study,TAT-Oct4 target gene was inserted into pET28a expression vector to construct pET28a-TAT-Oct4 recombinant expression vector. TAT-Oct4 fusion protein was mainly expressed with insoluble inclusion bodies in E.coli. The purity of expression product was more than90% after purification. And the average refolding field is 8.8% using urea gradient dialysis.Immunocytochemistry analysis showed that TAT-Oct4 recombinant protein could penetrate nearly100% cells membrane and was mainly concentrated in nucleus. The method of generating active TAT-Oct4 recombinant protein was established in this article. It provided not only effective study materials for cell reprogramming but also practical method for other reprogramming factors design.

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