Nickel-Induced Oligomerization of Proteins Containing 10-Histidine Tags

Immobilized metal ion affinity chromatography (IMAC) is an efficient method of protein purification. Several systems for protein expression and purification in which a recombinant protein is fused to an Nor C-terminal oligohistidine tag are commercially available. The tag of six to ten histidine residues binds with high affinity to metal-chelate complexes and allows for the rapid and facile purification of the fusion protein. Elution is carried out by lowering the pH or by the addition of competing metal ligands such as imidazole or EDTA (6). We have used IMAC to purify a homeodomain-containing protein for structural studies by nuclear magnetic resonance (NMR) spectroscopy. NMR requires rapid purification of micromole quantities of protein in good yield. The low molecular weight of the affinity tag and its ability to work under both native and denaturing conditions makes the polyhistidine tag IMAC the method of choice for purifying isotopically labeled proteins. The pre-B-cell leukemia transcription factor 1 (PBX1) gene was initially identified because of its involvement in some pre-B-cell leukemias (1,3). We have produced a fusion protein, Pbx233–319, using the pET vector system (Novagen, Madison, WI, USA), in which amino acids 233–319 of PBX are fused to an N-terminal ten-histidine tag by a Factor Xa cleavage site. The 108 amino acid (12 560 Da) protein, expressed in E. coli strain BL21 (λDE3), is purified on Ni2+-charged Chelating Sepharose Fast Flow (Amersham Pharmacia Biotech, Uppsala, Sweden) with a yield of approximately 40 mg of recombinant protein per liter of cell culture. Initial attempts at purifying Pbx233– 319 resulted in the apparent co-purification of a protein migrating at 41 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples incubated in elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM TrisHCl, pH 7.9) containing 6 M urea at 4°C for several days displayed a ladder of higher-molecular-weight proteins leading to the conclusion that Pbx233– 319 had oligomerized. This conclusion was confirmed by the reversible formation and dissociation of the oligomers by Ni2+ and EDTA. As shown in Figure 1, addition of EDTA converted the 41-kDa protein into monomeric Pbx233–319. Conversely, monomeric Pbx could be oligomerized to a higher-mass species by the addition of 10 mM nickel sulfate. In addition, it was found that while heating for a short period (<1 min) did not disrupt oligomerization, Pbx233– 319 could be returned to the monomeric form by vigorous boiling in loading buffer. We conclude that the polyhistidine affinity tag forms reversibly crosslinked oligomers that are resistant to 1% SDS, 6 M urea and 10 mM dithiothreitol (DTT). In samples without added nickel, we believe that sufficient metal leached off the IMAC affinity column for efficient oligomerization. Removal of the affinity tag by treatment with Factor Xa abolished the ability of Pbx233–319 to form oligomers (data not shown). We have also observed precipitation of Pbx233–319 during dialysis following IMAC or when using improperly prepared dialysis tubing. The precipitate dissolved upon addition of EDTA to the dialysis buffer. Similar observations have been reported for His-tag-purified yeast citrate synthase (2) and His-tagged eukaryotic transcription factors (5). The exact nature of the oligomers is unclear. Pbx233–319 migrates anomolously during SDS-PAGE as a 16-kDa band, and disulfide-linked dimers of Pbx233–319 migrate at 30 kDa (data not shown). In contrast, the nickel-induced oligomers showed apparent mobilities of 41 and 85 kDa, which suggest oligomer stoichiometries of 3 and 6, respectively. Aside from its intrinsic interest, understanding the nature of the metal-histidine complexes might be of interest for potential applications in cell surface receptor cross-linking, metaldependent enzyme-linked immunosorbent assays (ELISAs) (4) or metalsensing molecular switches. We chose to use a ten-histidine affinity tag to avoid problems of poor binding observed with other fusion protein constructs (T. Sprules, unpublished). Six-histidine affinity tags are more prevalent in the literature than ten-histidine tags but have lower affinity for the IMAC columns. The presence of ten histidine residues in our Pbx fusion protein may favor His-tag oligomerization. The presence of protein precipitates or oligomers after IMAC is of practical importance for subsequent chromatography. Samples appearing to be mono-