Purification of His-tagged proteins by immobilized chelate affinity chromatography: The benefits from the use of organic solvent

Recombinant proteins overexpressed in and purified from Escherichia coli co ntain impurities that are toxic in biological assays. The application of af finity purification procedures is often not sufficient to remove these toxi c components, We here describe a simple and fast, one-step protocol to remo ve these impurities highly efficiently. Four recombinant proteins were over expressed in E, coli as His-tagged fusion proteins and purified by immobili zed metal chelate affinity chromatography on Ni-NTA beads. Depending on the protein, the composition of the lysis buffer, and the washing protocol, va rious impurities appeared to be present in the purified protein preparation s. 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 remov al of proteins that aspecifically adhere to the beads or to the tagged prot ein, this procedure was particularly useful in removing endotoxins, Moreove r, we show that detergents such as NP-40, that are necessarily employed dur ing 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, end otoxins, and detergents. (C) 2000 Academic Press.