人体鼠双微体2C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 Human Oncoprotein MDM2 C-terminal-ZF&RING

作　　者：孟希[1] 陈婷[1] 张云龙[1] 黄旋[1] 刘晋源[1] 陆昌瑞 

机构地区：[1]东华大学化学化工与生物工程学院,上海201620

出　　处：《现代生物医学进展》2016年第27期5227-5231,共5页Progress in Modern Biomedicine

基　　金：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)

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Hi Trap 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。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 E.coli SUMO expression 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 E.coli. 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. Conclusions： 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.

关 键 词：鼠双微体2 C端结构域 蛋白表达纯化 单体结构 

分 类 号：Q816[生物学—生物工程] Q591