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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。
AIM: To construct and express the C-terminal domain of murine diabody 2 (MDM2) ZF & RING (aa.300-491) in Escherichia coli SUMO system, digest and purify to obtain the C-terminal domain of MDM2 protein Monomer structure, for its follow-up crystal research and MDM2 non-p53-dependent pathway to provide avenues. Methods: The zf & ring gene was recombined by using E. coli SUMO expression system. The constructed expression vector was expressed by induction, purified by Ni-NTA affinity chromatography, and analyzed by SDS-PAGE and Western blot. The purified fusion protein was digested with ULP1 to obtain the target protein ZF & RING, and the impurity DNA was tested and purified by Hi Trap Q FF ion exchange chromatography. Finally, the molecular structure of the protein was examined. Results: SUMO-ZF & RING recombinant vector was constructed. Recombinant vector highly efficient soluble expression in E. coli, purified and digested ZF & RING protein in the form of monomer. CONCLUSION: The ZF & RING protein of MDM2 C-terminal domain with high stability, high purity and monomer structure was successfully obtained by prokaryotic expression, purification, restriction enzyme digestion and chromatographic identification. It is a promising candidate for follow-up on MDM2, especially its non-p53 dependent pathway Structural and functional science provides ideas and ways.