Prokaryotic expression, purification and functional characterization of recombinant human RIP2
Prokaryotic expression, purification and functional characterization of recombinant human RIP2
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重组人RIP2的原核表达、纯化及功能表征
DOI：
10.1007/s11033-012-1995-z
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发表时间：
2013-01-01
影响因子：
2.8
通讯作者：
Xu, Donggang
中科院分区：
文献类型：
--
作者：
Cai, Xin;Wang, Min;Xu, Donggang
受体相互作用蛋白2(RIP2)是受体相互作用蛋白(RIP)家族的成员，在天然免疫和获得性免疫反应中发挥重要作用。RIP2的过表达介导了不同的信号通路，包括核因子-kappaB的激活和细胞死亡。为了进一步研究RIP2的体外生物学活性，需要大量的纯化蛋白。为此，从人Ramos(人Burkitt淋巴瘤)细胞中克隆了RIP2的全长基因，并将其克隆到原核表达载体pET22b中，转化入大肠杆菌BL21(DE3)感受态细胞。IPTG诱导RIP2的表达。SDS-PAGE分析表明，重组人RIP2(RhRIP2)主要以可溶性组分形式在细胞裂解液上清液中表达。重组蛋白随后被His Trap FF粗提物纯化到90%的纯度。四甲基偶氮唑盐比色实验表明，纯化的重组人RIP2在不同的细胞系中具有不同的功能差异，对MCF7细胞有特异性的抑制作用，但对Ramos细胞的增殖有促进作用。此外，我们还发现，RIP2可以抑制MCF7细胞中规范的NF-kappa B的激活，而激活Ramos细胞中的非规范的NF-kappa B信号，这些数据表明RIP2在体外参与了不同的信号通路，从而发挥了其特异性作用。我们的研究结果为进一步探讨RIP2在肿瘤发生中的调控机制提供了新的线索。
Receptor-interacting protein 2 (RIP2) is a member of the receptor interacting protein (RIP) family and plays an important role in the innate and adaptive immune responses. Overexpression of RIP2 mediates divergent signaling pathways including NF-kappa B activation and cell death. To further investigate the biological activity of RIP2 in vitro, a large amount of purified protein is required. For this purpose, the full length of RIP2 was cloned from human Ramos (human Burkitt lymphoma) tumor cells and inserted in a prokaryotic expression vector pET22b, and then the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells. The expression of RIP2 was induced with IPTG. SDS-PAGE analysis showed that recombinant human RIP2 (rhRIP2) was mainly expressed as soluble fraction in the supernatant of the cell lysate. The recombinant protein was subsequently purified by His Trap FF crude to a purity of 90 %. MTT assay of the purified rhRIP2 showed its functional diversity in different cell lines, a specific inhibitory effect on MCF7 cells, but a promotion on the proliferation of Ramos cells. Furthermore, we identified that rhRIP2 could suppress activation of canonical NF-kappa B in MCF7 cells and activate non-canonical NF-kappa B signaling in Ramos cells, these data suggested that RIP2 participates in different signaling pathways contributing to its specific effects in vitro. Our results provided new clues to further explore the regulation mechanisms of RIP2 in tumorigenesis.