High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli.
High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli.
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天然人表皮生长因子在大肠杆菌中的高效表达、纯化和功能表征
DOI：
10.1155/2016/3758941
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发表时间：
2016
影响因子：
--
通讯作者：
Wang J
中科院分区：
文献类型：
--
作者：
Ma Y;Yu J;Lin J;Wu S;Li S;Wang J
人表皮生长因子(HEGF)是一种促进多种细胞增殖的小分子有丝分裂生长多肽，广泛应用于临床。然而，由于单体hEGF中的三个二硫键使其不能在体内折叠成正确的三维结构，因此天然hEGF在大肠杆菌中的高效表达一直没有成功。为了解决这个问题，我们在hEGF的C端融合了MXE Gyra内含素(MXE)，然后是小泛素相关修饰物(SUMO)和10倍的His-Tag，构建了hEGF-MXE-SUMO-H10嵌合蛋白。融合蛋白的表达浓度为281 mg/L，占菌体总可溶性蛋白的59.5%。融合蛋白经亲和层析纯化后，经硫醇诱导的N端切割可释放29.4 mg/L的天然人表皮生长因子，且无任何酶促反应。用四甲基偶氮唑蓝(四甲基偶氮唑蓝)比色法检测商业和重组hEGF对Balb/c 3T3细胞有丝分裂活性的影响，结果表明，重组hEGF蛋白刺激细胞增殖的作用与商业蛋白相似。本研究显着提高了重组大肠杆菌系统中hEGF的产量，降低了成本，为药物开发提供了一种更好的原生hEGF生产策略。
Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.
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影响因子：
4.7
作者：
Sydor, JR;Mariano, M;Nock, S
通讯作者：
Nock, S
影响因子：
4.4
作者：
Sivakesava, S;Xu, ZN;Wong, WKR
通讯作者：
Wong, WKR
影响因子：
1.6
作者：
Esipov, Roman S.;Stepanenko, Vasily N.;Miroshnikov, Anatoly I.
通讯作者：
Miroshnikov, Anatoly I.
影响因子：
1.6
作者：
Su, Zhijian;Huang, Yadong;Li, Xiaokun
通讯作者：
Li, Xiaokun
DOI：
10.1152/ajpendo.00390.2002
发表时间：
2003-04-01
影响因子：
5.1
作者：
Kishi, A;Nakamura, T;Kashiwagi, A
通讯作者：
Kashiwagi, A