| 孟希 陈婷 张云龙 黄旋 刘晋源 陆昌瑞△.人体鼠双微体2 C端结构域ZF&RING 重组蛋白的表达、纯化及单体结构研究[J].现代生物医学进展英文版,2016,16(27):5227-5231. |
| 人体鼠双微体2 C端结构域ZF&RING 重组蛋白的表达、纯化及单体结构研究 |
| The Expression, Purification and Monomer Structure Research of HumanOncoprotein MDM2 C-terminal -ZF&RING* |
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| DOI： |
| 中文关键词: 鼠双微体2 C 端结构域 蛋白表达纯化 单体结构 |
| 英文关键词: MDM2 C-terminus Protein expression and purification Monomeric form |
| 基金项目:国家自然科学基金项目（31300603）；上海特别委任教授(东方学者)项目（2012-28）；
中央高校基本科研业务费专项资金资助项目（14D110511）；国家大学生创新性实验计划项目（14T10501） |
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| 中文摘要: |
| 目的：通过在大肠杆菌SUMO 系统中对鼠双微体2 (MDM2) C端结构域ZF&RING (aa.300-491)进行构建并进行表达，酶切
和纯化，从而得到MDM2 蛋白C 端结构域的单体结构，为其后续的晶体研究及MDM2 非p53 依赖途径的研究提供途径。方法：
利用大肠杆菌SUMO 表达系统对zf&ring 基因进行重组构建。构建成功的表达载体经诱导表达优化后，通过Ni-NTA进行亲和层
析纯化，并利用SDS-PAGE 及Western blot 鉴定分析。纯化后的融合蛋白经ULP1 酶切得到目的蛋白ZF&RING，并通过HiTrap
Q FF离子交换层析检验和去除杂质DNA。最后通过分子筛检验其蛋白结构。结果：构建了SUMO-ZF&RING重组载体。重组载
体在大肠杆菌高效可溶性表达，纯化并酶切后的目的蛋白ZF&RING以单体形式存在。结论：通过原核表达、纯化、酶切及层析发
鉴定，成功获得高稳定、高纯度且为单体结构的MDM2 C端结构域ZF&RING蛋白，为后续关于MDM2，尤其是其非p53 依赖途
径的结构学和功能学提供了思路和途径。 |
| 英文摘要: |
| Objective:To present a new method to recombinantly express and purify the C-terminal Zinc Finger and RING domain
of MDM2 (ZF&RING) in monomeric form, thus providing a new avenue for characterizing MDM2 p53-independent roles through future
structure and function studies.Methods:ZF&RING gene was first constructed in SUMOexpression system. The expression vectors
containing recombinant genes were purified by Ni-NTA affinity chromatography and analyzed by SDS-PAGE and western blot after optimized
expressing in . The SUMO tag and DNA was removed by protease ULP1 and ion-exchange. The aggregation state of the purified
ZF&RING was finally confirmed by size exclusion chromatography.Results:SUNO-zf&ring recombinant genes were constructed
and the fusion protein highly soluble expressed in E.coli. After well purified and digested, the target ZF&RING was validated to be
monomeric by Gel filtration chromatography.Conclusion:Through prokaryotic expression, purification and digestion, we successfully
obtained human oncoprotein MDM2 C-terminal domain ZF&RING monomeric proteins with high stability and purity under physiological
conditions. The purified protein could be used for further studies of structure and function relationships of MDM2 in p53-independent
pathways. |
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