Purification of His-Tagged Proteins by Immobilized Chelate Affinity Chromatography: The Benefits from the Use of Organic Solvent Kees L. M. C. Franken,* Hoebert S. Hiemstra,* Krista E. van Meijgaarden,* Yanri Subronto,* J. den Hartigh,† Tom H. M. Ottenhoff,* and Jan W. Drijfhout* *Department of Immunohematology and Blood Bank and †Department of Clinical Pharmacy, Leiden University Medical Center, 2300 RC Leiden, The Netherlands Received August 17, 1999, and in revised form October 13, 1999 Recombinant proteins overexpressed in and puri- fied from Escherichia coli contain impurities that are toxic in biological assays. The application of affinity purification procedures is often not sufficient to re- move these toxic components. We here describe a sim- ple and fast, one-step protocol to remove these impu- rities highly efficiently. Four recombinant proteins were overexpressed in E. coli as His-tagged fusion pro- teins and purified by immobilized metal chelate affin- ity 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 re- moving endotoxins. Moreover, we show that deter- gents 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 prod- ucts contain significantly less contaminating E. coli proteins, endotoxins, and detergents. © 2000 Academic Press For the purification of recombinant proteins, various classical separation procedures can be employed, which are based on the physicochemical properties of the protein, such as charge, size, and hydrophobicity. These procedures are often time consuming and labo- rious. Consequently, simple and rapid alternatives for purification have been developed, which make use of a particular property of an amino acid sequence that is fused to the recombinant protein as affinity tag. Exam- ples of techniques exploiting this strategy are: (a) the glutathione S-transferase fusion system used in com- bination with glutathione–Sepharose beads (1), (b) the maltose-binding protein fusion used in combination with maltose beads (2), (c) the chitin-binding protein fusion used in combination with chitin beads (3), (d) the protein A fusion system combined with various immunoglobulin columns (4), (e) epitope tagging used together with specific antibodies (5), (f) biotin tags used in combination with avidin–agarose (6), and (g) the histidine-tagging techniques for use in metal chelate affinity chromatography (7–14). In contrast to most other procedures, the advantage of histidine tagging is that it can be employed also under denaturing condi- tions (15). Although the interactions of the tag with the affinity material are highly specific, contaminating proteins as well as other nonprotein contaminants can bind aspe- cifically to the beads or to the tagged protein of interest itself. Here we report that by using organic solvent washing steps, the presence of impurities in the tagged end products is significantly reduced. Results will be reported for four different proteins: T5 (19.6 kDa) from Mycobacterium leprae (16), TonB (31.6 kDa) from Hae- mophilus influenza (17,18), ferritin (23.8 kDa) from Mycobacterium tuberculosis (19), and E7 (14.2 kDa) from human papillomavirus serotype 16 (20). In addition to the removal of protein impurities we were also particularly interested in removing Esche- richia coli endotoxins, since these are known to inter- fere with several cellular assays, e.g., by activating phagocytic cells and by inhibiting lymphocytic re- sponses. Although endotoxins can be removed by ap- plication of a number of chromatographic columns, such as Detoxigel (Pierce), a major limitation is that Protein Expression and Purification 18, 95–99 (2000) doi:10.1006/prep.1999.1162, available online at http://www.idealibrary.com on 95 1046-5928/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.