Immobilized metal affinity chromatography (IMAC) is one of the most common techniques for purifying histidine-tagged proteins (His-tagged proteins), in which hexahistidine (His6) is generally used as a histidine tag. IMAC relies on coordination bonds between a His-tag and transition metal ions (such as Ni2+, Co2+, and Cu2+) immobilized on matrices in columns [1,2]. In nature, there are few histidine-rich proteins; specifically, the content of histidine residues in globular proteins is approximately 2%, almost half of which is inside the proteins [3]. Accordingly, IMAC can selectively separate His-tagged proteins, leading to high-purity purification; in addition, it has advantages in terms of cost performance [4,5]. It should also be noted that His-tags have little effect on target protein structures [6] and functions because of their short length (generally, 6 residues) [7]. However, IMAC requires chelating agents, low-pH solutions, and high-concentration imidazole solutions for eluting His-tagged proteins from the columns. Chelating agents (such as ethylenediaminetetraacetic acid (EDTA)) reduce the protein-binding capacity of IMAC resins because they remove metal ions from the resins. Low-pH solutions can induce undesirable protein denaturation and hence undermine protein functions. Imidazole, which can remain even after purification, often affects analytical results such as NMR spectra [8], [9], [10], inhibits enzyme activities [9,11,12], and possibly induces precipitation of His-tagged proteins in freezing preservation [9,13]. Furthermore, a small number of metal ions on the matrices can be eluted together with His-tag proteins even without using chelating agents [14,15]. The leakage of metal ions leads to a decrease in the purification yield of His-tagged proteins; in addition, Ni2+ and Co2+ which are often used in IMAC are considered carcinogens [16]. Thus, IMAC has several challenges to overcome for facilitating His-tagged protein applications.

Ion exchange chromatography (IEX) is often used along with IMAC for the purification of His-tagged proteins [17], [18], [19], [20]. Since IEX is a method for protein purification based on the electrostatic interaction of proteins with the charged ligands on the matrices, it primarily depends on the concentration of ions in the mobile phase. Accordingly, IEX can be utilized to purify His-tagged proteins using only common salts (but not chelating agents, imidazole, and heavy metal ions). However, electrostatic interactions acting on the IEX have lower selectivity for His-tagged proteins, compared to coordination bonds in IMAC. Therefore, IEX is not often used for one-step purification of His-tagged proteins (in contrast to IMAC [11,21]) and is alternatively used as upstream and downstream processes in addition to IMAC [17], [18], [19], [20].

Phosphate-modified zirconia (ZrO2-P) particles are used as cation exchangers for protein purification [22], [23], [24]. The materials were developed and applied to protein purification in 1990s [23]. The modification of zirconia particles with phosphate groups was used to improve the selectivity for proteins. The use of zirconia for protein purification is reasonable because zirconia is a human-friendly (biocompatible) material [25,26]. Zirconia particles have advantages over organic matrices, such as high chemical stability, high-temperature tolerance, and high-pressure resistance, because of their inorganic nature. The high chemical stability and high-temperature tolerance enable cleaning and regeneration of the particles under harsh conditions using chemicals such as acid, basic, and detergents and using heat treatment; the high-pressure resistance enables rapid purification of target proteins on a chromatographic column. It was reported that ZrO2-P particles selectively adsorb immunoproteins [27], [28], [29], [30], and hence they can be used for isolating them with high purity. The immunoprotein-adsorption ability of ZrO2-P particles can be controlled by varying the concentration of salts at neutral pH [29,30]. Because immunoproteins can be purified under such mild conditions, they can maintain their activity [29,30]. In a previous study, it was suggested that the immunoglobulin-adsorption of ZrO2-P particles is governed by the electrostatic interaction between phosphate groups on the zirconia particles and basic amino acid residues (i.e., His, Lys, and Arg) which exist in the CL–CH1–hinge region of immunoglobulin [31]. The electrostatic interaction also works in the interplay between the ZrO2-P particles and basic homopeptides, including oligohistidine; the longer the basic homopeptides, the more strongly they are adsorbed onto the ZrO2-P particles [31].

Here, it was assumed that the oligohistidine-adsorption ability of the ZrO2-P particles can be applied for the purification of His-tagged proteins. On the basis of the above mechanism, His-tagged proteins should be adsorbed onto ZrO2-P particles at neutral pH and desorbed from them at high concentrations of salts. This purification using ZrO2-P particles will overcome the disadvantages of current purifications using IMAC columns, such as the necessity of using low pH solutions, chelating agents, imidazole, and heavy metals. In addition, since purification using ZrO2-P particles requires only salt solutions as eluents [23,29,30], it will be cost-effective.

The present study investigated the His6-adsorption ability of ZrO2-P particles and tested the purification of His-tagged (strictly His6-tagged) proteins using a column filled with ZrO2-P particles (hereafter referred to as the ZrO2-P column), where His6 was used as a model of the His-tag moiety. Two cation exchangers were used as reference materials as follows: cross-linked agarose beads modified carboxymethyl groups (CM Sepharose) as a representative of weak cation exchangers and cross-linked agarose beads modified with sulfopropyl groups (SP Sepharose) as a representative of strong cation exchangers. The purification using the ZrO2-P column was performed for two model proteins—His-tagged green fluorescent protein (GFP–His) and His-tagged alkaline phosphatase fused with maltose binding protein (His-MBP-PhoA) (Fig. S1). The results showed that His6 and the His-tagged proteins are more strongly adsorbed onto the ZrO2-P particles than onto the other cation exchangers at neutral pH. The purity of the His-tagged proteins with the ZrO2-P column was almost comparable to that obtained with a conventional IMAC column (Ni-NTA column). The present findings suggest the possibility that the ZrO2-P column can be used for His-tagged protein purification as an alternative to an IMAC column.