Bacterial expression and purification of biologically active human TFF2

CORC > 昆明动物研究所 > 昆明动物研究所 > 动物模型与人类重大疾病机理重点实验室 > 动物活性蛋白多肽组学

	Bacterial expression and purification of biologically active human TFF2
	Zhuang YH 1,2; Li SM 1,3; Zou H[]4; Lee WH[]1; Yu GY 1,3; Zhang Y 1; Xiang Y 1
刊名	ZOOLOGICAL RESEARCH
	2012
卷号	33 期号:2 页码:144-150
关键词	Wound healing Anti-apoptosis Cell migration Expression TFF2
其他题名	人 TFF2 基因的原核表达与纯化
通讯作者	newzouhao@163.com
合作状况	其它
中文摘要	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 °C, 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 5 mg/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 50 μmol/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。
英文摘要	Human trefoil factor 2 (hTFF2) is considered as one of the most important initiators of mucosal healing in the gastrointestinal tract by promoting cell migration and suppressing apoptosis. However, it is hard to obtain hTFF2 from human tissue and many recombinant hTFF2 produced in vitro exist as fusion proteins. The purpose of the present study was to produce native hTFF2 while maintaining its biological activities. The open reading frame of hTFF2 was inserted into a pET-32a(+) expression vector, and hTFF2-TRX fusion protein was successfully expressed in Escherichia coli and purified by Nickel-nitrilotriacetic acid affinity chromatography and reverse-phase HPLC steps. The recombinant fusion protein (purity>95%) was cleaved by Factor Xa at 23 Degrees Celsius to release hTFF2. After removal of Factor Xa and undigested fusion proteins, hTFF2 was purified and identified by SDS-PAGE and Western blotting. The yield of recombinant hTFF2 was about 5 mg/L. The recombinant hTFF2 could promote IEC-6 cells migration and in vitro wound healing via the activation of ERK1/2. Recombinant hTFF2 could also inhibit apoptosis of HCT-116 cells induced by 50 μmol/L ceramide. In summary, our results showed that the recombinant hTFF2 was expressed in E. coli and successfully purified after cleavage with the fusion partner with high yield while maintaining its biological activities. Recombinant hTFF2 might be useful for investigating the molecular mechanism of hTFF2 and development of hTFF2-related drugs.
收录类别	其他
资助信息	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)
语种	英语
内容类型	期刊论文
源URL	[http://159.226.149.26:8080/handle/152453/9584]
专题	昆明动物研究所_动物活性蛋白多肽组学昆明动物研究所_动物模型与人类重大疾病机理重点实验室
作者单位	1.Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology,Kunming Yunnan 650223, China 2.Department of Neurosurgery, Yunnan No.2 People’s Hospital, Kunming Yunnan 650021, China; 3.Department ofBiochemistry, Kunming Medical College, Kunming Yunnan 650500, China 4.Department of Hepatobiliary Surgery, the SecondAffiliated Hospital of Kunming Medical College, Kunming Yunnan 650101, China
推荐引用方式 GB/T 7714	Zhuang YH,Li SM,Zou H[*],et al. Bacterial expression and purification of biologically active human TFF2[J]. ZOOLOGICAL RESEARCH,2012,33(2):144-150.
APA	Zhuang YH.,Li SM.,Zou H[].,Lee WH[].,Yu GY.,...&Xiang Y.(2012).Bacterial expression and purification of biologically active human TFF2.ZOOLOGICAL RESEARCH,33(2),144-150.
MLA	Zhuang YH,et al."Bacterial expression and purification of biologically active human TFF2".ZOOLOGICAL RESEARCH 33.2(2012):144-150.