Purification of His-tagged proteins by immobilized chelate affinity chromatography: The benefits from the use of organic solvent
Purification of His-tagged proteins by immobilized chelate affinity chromatography: The benefits from the use of organic solvent
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DOI：
10.1006/prep.1999.1162
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发表时间：
2000-02-01
影响因子：
1.6
通讯作者：
Drijfhout, JW
中科院分区：
文献类型：
--
作者：
Franken, KLMC;Hiemstra, HS;Drijfhout, JW
在大肠杆菌中过表达和纯化的重组蛋白含有在生物测定中有毒的杂质。亲和纯化程序的应用通常不足以去除这些有毒成分，我们在这里描述了一种简单快速的一步方案来高效地去除这些杂质。四个重组蛋白在大肠杆菌中过量表达为His-标记的融合蛋白，并通过在Ni-NTA珠上的固定化金属螯合物亲和层析纯化。根据蛋白质、裂解缓冲液的组成和洗涤方案，纯化的蛋白质制剂中似乎存在各种杂质。在这里，我们展示了在洗涤步骤期间使用60%异丙醇如何从最终产物中去除大部分这些污染物。除了去除特异性粘附于珠或标记蛋白的蛋白质之外，该程序在去除内毒素方面特别有用。此外，我们展示了在裂解期间必须使用的去污剂如NP-40也被有效地去除。最后，我们表明蛋白质在异丙醇处理后能够正确地重折叠。因此，所得最终产物含有显著更少的污染性E。大肠杆菌蛋白、内毒素和去污剂。(C)北京大学出版社.
Recombinant proteins overexpressed in and purified from Escherichia coli contain impurities that are toxic in biological assays. The application of affinity purification procedures is often not sufficient to remove these toxic components, We here describe a simple and fast, one-step protocol to remove these impurities highly efficiently. Four recombinant proteins were overexpressed in E, coli as His-tagged fusion proteins and purified by immobilized metal chelate affinity chromatography on Ni-NTA beads. Depending on the protein, the composition of the lysis buffer, and the washing protocol, various impurities appeared to be present in the purified protein preparations. Here we show how the use of 60% isopropanol during washing steps removed most of these contaminants from the end products, In addition to the removal of proteins that aspecifically adhere to the beads or to the tagged protein, this procedure was particularly useful in removing endotoxins, Moreover, we show that detergents such as NP-40, that are necessarily employed during lysis, are also efficiently removed, Finally, we show that proteins are able to refold correctly after isopropanol treatment. Thus, the resulting end products contain significantly less contaminating E. coli proteins, endotoxins, and detergents. (C) 2000 Academic Press.