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Bacterial expression and purification of biologically active human TFF2

ZHUANG Yong-Hui LI Si-Man YU Guo-Yu ZHANG Yong XIANG Yang ZOU Hao LEE Wen-Hui

ZHUANG Yong-Hui, LI Si-Man, YU Guo-Yu, ZHANG Yong, XIANG Yang, ZOU Hao, LEE Wen-Hui. Bacterial expression and purification of biologically active human TFF2. Zoological Research, 2012, (2): 144-150. doi: 10.3724/SP.J.1141.2012.02144
Citation: ZHUANG Yong-Hui, LI Si-Man, YU Guo-Yu, ZHANG Yong, XIANG Yang, ZOU Hao, LEE Wen-Hui. Bacterial expression and purification of biologically active human TFF2. Zoological Research, 2012, (2): 144-150. doi: 10.3724/SP.J.1141.2012.02144


doi: 10.3724/SP.J.1141.2012.02144
基金项目: “973”项目(2010CB529800); 国家基金委面上项目(81160302, 30870304); 中国科学院“西部之光”(Y102291081)
  • 中图分类号: Q786; Q251

Bacterial expression and purification of biologically active human TFF2

Funds: This work was supported by grants from the National Basic Research Program of China (973 Program, 2010CB529800), the Chinese National Natural Science Foundation (81160302, 30870304), the “Western Light Project” from the Chinese Academy of Sciences (Y102291081), and the Science and Technology Department of Yunnan Province (2011C1139)
  • 摘要: 人三叶因子2(hTFF2)具有促进细胞迁移和抑制细胞凋亡的活性,所以被认为是胃肠黏膜修复的启动者之一。因为从人组织中获得hTFF2比较困难,而且体外产生的重组hTFF2大都以融合蛋白的形式存在,所以该研究的目的是在体外产生不带任何融合蛋白的游离型hTFF2。hTFF2的开放阅读框被插入pET-32a(+)表达载体,然后在大肠杆菌中表达出带有硫氧还蛋白融合部分的hTFF2融合蛋白。进而利用融合蛋白的组氨酸标签使用镍亲和色谱柱以及反向高压色谱柱对目的蛋白进行纯化。23℃,FXa因子裂解纯度高达95%的融合蛋白以得到游离型hTFF2。在去除FXa因子和尚未被切开的融合蛋白后,获得的游离型hTFF2被SDS-PAGE和Western blotting所证实。重组游离型hTFF2的产量约为5mg/L,并且hTFF2能促进IEC-6细胞的迁移以及体外的伤口修复,而这些活性是依赖于ERK1/2的激活。同时,hTFF2也能抑制50μmol/L神经鞘氨醇所引起的HCT-116细胞的凋亡。总之,研究结果表明,在大肠杆菌中高产量地成功表达出具有生物学活性的游离型hTFF2,这为研究TFF2的分子机制,以及研制和开发TFF2的相关药物都提供很大的帮助。
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  • 收稿日期:  2011-12-14
  • 修回日期:  2012-02-22
  • 刊出日期:  2012-04-22