Xiang Hui, Wynn Richard, Nguyen Lien-Hanh T, Ross O Harold, Ahrens Douglas P, O'Neil Karyn T, Hollis Gregory F, Patrick Denis R
J Chromatogr A. 2002 Nov 29;978(1-2):153-64. doi: 10.1016/s0021-9673(02)01429-2.
Undesired adsorption of host cell proteins poses a big challenge for immobilized metal-ion affinity chromatography (IMAC) purification. In this study, by using His6-tagged protein Fab OPG C11 from Escherichia coli fermentation as a model, we found that the presence of low concentrations of EDTA-Mg2+ in feed streams weakens the adsorption but makes it more specific towards polyhistidine tag. By combining EDTA-Mg2+ treatment and periplasmic extraction, we developed a one-step purification procedure for His6-tagged recombinant Fab OPG C11 using Ni-IDA (iminodiacetic acid) chromatography. This procedure eliminated the buffer exchange step after periplasmic extraction, which is usually required before IMAC in order to remove EDTA. In addition to savings on time and cost, this procedure eliminates undesired adsorption of most host cell proteins thus significantly improves the purity of polyhistidine-tagged recombinant proteins. The strategy of EDTA-Mg2+ treatment may have general application potentials.
宿主细胞蛋白的非特异性吸附对固定化金属离子亲和色谱（IMAC）纯化构成了巨大挑战。在本研究中，以来源于大肠杆菌发酵的His6标签蛋白Fab OPG C11为模型，我们发现进料流中低浓度EDTA-Mg2+的存在会减弱吸附，但会使其对多组氨酸标签的吸附更具特异性。通过结合EDTA-Mg2+处理和周质提取物，我们开发了一种使用镍-亚氨基二乙酸（Ni-IDA）色谱对His6标签重组Fab OPG C11进行一步纯化的方法。该方法省去了周质提取物后通常在IMAC之前进行的用于去除EDTA的缓冲液交换步骤。除了节省时间和成本外，该方法还消除了大多数宿主细胞蛋白的非特异性吸附，从而显著提高了多组氨酸标签重组蛋白的纯度。EDTA-Mg2+处理策略可能具有广泛的应用潜力。
