Size of Protein is a Major Factor that Affects Retention on Preparative IMAC Columns

Immobilized metal affinity chromatography (IMAC) is a specific high-capacity technique used in large-scale purification of proteins. IMAC exploits the ability of immobilized metal ions to form coordination bonds with atoms in the side chains of certain amino acids. The technique is generally robust. However, several factors still affect column binding capacity, retention, yield and purity of proteins during IMAC. It was observed that the recovery of 6× histidine, (His)₆-tagged proteins from metal affinity columns differ significantly depending on the size of the protein. To test this observation, we determined the effect of protein size, flow-rate, number and position of (His)₆ tag on the retention of highly expressing proteins on commercial Ni²⁺ and Co²⁺ IMAC columns. All experiments were performed in phosphate buffer to eliminate interference of amine-containing buffers with the binding of the (His)₆ tag to the columns. Column retention was determined as the ratio of protein of interest in the supernatant (input) to flow-through (output). Data obtained suggest that regardless of the flow-rate, (His)₆ tag position and number, the size of protein is a major factor affecting column retention and therefore recovery during column IMAC purification. Small and medium-sized proteins (~ 50 kDa) have higher column retention than bigger proteins, resulting in higher recovery. These outcomes provide important information to consider when performing IMAC.