Epitope imprinting enhanced IMAC (EI-IMAC) for highly selective purification of His-tagged protein
Epitope imprinting enhanced IMAC (EI-IMAC) for highly selective purification of His-tagged protein
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表位印迹增强 IMAC (EI-IMAC) 用于高选择性纯化 His 标记蛋白
DOI：
10.1039/c5tb02505b
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发表时间：
2016-01-01
影响因子：
7
通讯作者：
Zhang, Yukui
中科院分区：
文献类型：
--
作者：
Li, Senwu;Yang, Kaiguang;Zhang, Yukui
重组蛋白技术在生物制药、蛋白质组学、结构与功能生物学等领域占有重要地位。然而，his标记蛋白的纯化受到杂质的严重阻碍，这是重组蛋白的大部分。这些杂质，包括宿主蛋白固有的富含半胱氨酸和组氨酸的区域或金属中心，通常超出了常用IMAC材料的纯化能力。为了消除这一屏障，利用his标记蛋白的共同末端His-tag作为模板，通过表面表位印迹提高IMAC的选择性，开发了一种新的纯化材料。通过TEM、热重分析、x射线光电子能谱、DLS尺寸和zeta电位测量等表征来证明印迹壳的制备。结果显示高印迹因子为7.1。吸附动力学不受表面印迹壳的影响，可在15 min内达到吸附平衡。与底物IMAC相比，新型表位印迹增强IMAC （EI-IMAC）对粗细胞裂解中his标记重组蛋白的选择性有明显提高（纯度提高5%）。
Recombinant protein technology occupies an important position in fields including biopharmaceutics, proteomics, structural and functional biology. However, the purification of His-tagged protein, the majority portion of recombinant protein, is seriously hindered by impurities. These impurities, including host proteins with inherent cysteine and histidine-rich regions or metal centers, are usually beyond the purification ability of commonly used IMAC materials. To remove this barrier, a novel purification material was developed through enhancing the selectivity of IMAC by means of surface epitope imprinting using His-tag, the common terminal of His-tagged protein, as the template. Characterizations including TEM, thermogravimetric analysis, X-ray photoelectron spectroscopy, measurement of DLS size and zeta potential were carried out to prove the fabrication of the imprinted shell. Results exhibited a high imprinting factor of 7.1. Besides, the adsorption kinetics were not affected by the surface imprinted shell and could reach adsorption equilibrium within 15 min. Compared with the substrate IMAC, the novel epitope imprinting enhanced IMAC (EI-IMAC) showed an obvious improvement (5% increase of purity) in the selectivity of His-tagged recombinant protein from crude cell lysis.