Protein Expression and Purification 21, 352–360 (2001) doi:10.1006/prep.2000.1384, available online at http://www.idealibrary.com on Copurification of the Lac Repressor with Polyhistidine- Tagged Proteins in Immobilized Metal Affinity Chromatography Ro ´isı ´n M. Owens, Andrew Grant, Nicholas Davies, and C. David O’Connor 1 Division of Biochemistry and Molecular Biology, University of Southampton, Bassett Crescent East, SO16 7PX, United Kingdom Received November 8, 2000, and in revised form December 6, 2000 available coordination sites arranged in an octahedral One of the commonly used resins for immobilized configuration around each metal atom. Each of these metal affinity purification of polyhistidine-tagged coordination sites has the capacity to interact with an recombinant proteins is TALON resin, a cobalt (II)– electron-rich ligand such as histidine, tryptophan, or carboxymethylaspartate-based matrix linked to Seph- cysteine. arose CL-6B. Here, we show that TALON resin Histidine residues in particular exhibit highly selec- efficiently purifies the native form of Lac repressor, tive coordination with certain transition metals and as which represents the major contaminant when (His) 6 - such have great utility in IMAC, especially due to the tagged proteins are isolated from Escherichia coli host fact that histidine binds under conditions of physiologi- cells carrying the lacI q gene. Inspection of the crystal cal pH. This observation has been exploited to allow structure of the repressor suggests that three His resi- the selective purification of recombinant proteins that dues (residues 163, 173, and 202) in each subunit of the have been engineered to contain a stretch of His resi- tetramer are optimally spaced on an exposed face of dues, typically at the N- or C-terminus (3). The presence the protein to allow interaction with Co(II). In addition of four to six contiguous histidine residues engineered to establishing a more efficient procedure for purifica- into a tag generates a ligand that can stably bind to a tion of the Lac repressor, these studies indicate that metal chelate affinity column and withstand extensive non-lacI q -based expression systems yield significantly purer preparations of recombinant polyhistidine- washing to remove nonspecifically bound proteins. tagged proteins.  2001 Academic Press However, the histidine residues need not of necessity be consecutive. It has been reported that proteins dis- playing single histidinyl side chains on their surfaces may in some instances be resolved by IMAC (4). Immobilized metal affinity chromatography (IMAC) 2 Several of the resins available for IMAC of polyhistid- is a versatile generic method for purifying proteins to ine-tagged proteins use Ni 2+ , e.g., Ni 2+ –nitrilotri- near homogeneity in a single step (1, 2). The method acetate, and can capture tagged proteins even under is based on the observation that proteins fused to poly- strongly denaturing conditions (5). However, such res- histidine tags bind with relatively high affinity to elec- ins suffer from metal loss and quite stringent conditions tropositive transition metals, including Co 2+ , Ni 2+ , Cu 2+ , are generally required to elute proteins following bind- and Zn 2+ . Metals such as cobalt and nickel have six ing. Nickel-based IMAC resins have also been reported to bind host cell proteins containing exposed histidine 1 To whom correspondence should be addressed at Division of Bio- residues (6), leading to a decrease in the efficiency of chemistry and Molecular Biology, School of Biological Sciences, Uni- protein purification. To circumvent these problems, versity of Southampton, Bassett Crescent East, Southampton SO16 a cobalt (II)–carboxymethylaspartate-based matrix 7PX, UK. Fax: +44 (0)2380 594459. E-mail: doc1@soton.ac.uk. linked to Sepharose CL-6B, TALON resin, has been 2 Abbreviations used: IMAC, immobilized metal affinity chromatog- raphy; LB, Luria broth; IPTG, isopropyl--D-thiogalactoside. introduced (8), which has been reported to exhibit better 352 0091-7435/01 $35.00 Copyright  2001 by Academic Press All rights of reproduction in any form reserved.