Chromatographic purification of histidine-tagged proteins using zirconia particles modified with phosphate groups

Immobilized metal ion affinity chromatography (IMAC) is one of the most common purification techniques for histidine-tagged proteins (His-tagged proteins). IMAC enables the purification of His-tagged proteins at high purity on the basis of coordination bonds between His-tags and metal ions (such as Ni2+, Co2+, and Cu2+) immobilized on the matrices in columns. However, IMAC requires low-pH solutions or high-concentration imidazole solutions for eluting His-tagged proteins, which can affect protein conformation and activity. The present study provides a His-tagged protein purification method using zirconia particles modified with phosphate groups. This method is based on the electrostatic attractions between a His-tag moiety of proteins and phosphate groups on the zirconia particles; this method requires only high-concentration salt solutions at pH 7.0 for eluting the proteins. A column packed with phosphate-modified zirconia particles was demonstrated to enable the purification of two model His-tagged proteins-His-tagged green fluorescent protein and His-tagged alkaline phosphatase fused with maltose binding protein. Thus, this chromatography method is useful for purifying His-tagged proteins without any pH stress or additives. Additionally, because of the mechanical properties of the zirconia particles, this technique enables high-performance purification at a high flow rate.