Immobilized Metal Ion Affinity Chromatography and Its Application

Abstract

Immobilized metal ion affinity chromatography (IMAC) is a group-specific affinity separation technique, which is based on specific interactions between molecules in solution and metal ions fixed to a solid support. Metal ions, being electron acceptors, react with ligands and electron-donor groups of biomolecules. In biomolecules, electron donors are surface-exposed atoms of nitrogen, sulfur and oxygen, potentially also phosphorus. As biomolecules (e.g. proteins) are bound to metal chelates, weakly bound ligands (e.g. water) are displaced from the metal chelate complex. Elution of the target proteins is achieved mostly by protonation (lowering pH) or by addition of a competing agent (e.g. imidazole). The IMAC with Co²⁺ relies on the formation of weak coordination bonds between Co²⁺ ions and basic groups of proteins, in particular histidine residues. The use of the IMAC with Co²⁺ shows distinct advantages such as high loading, mild elution conditions, simple regeneration and low cost. The technique is therefore used for various purposes, including preparative and analytical purification of proteins and study of their surface topography. Efficient purification of recombinant proteins with engineered histidine affinity tags attached to the N- or C- terminus is another important application of this method.