Immobilized metal ion affinity chromatography of histidine-tagged fusion proteins

Abstract

Immobilized metal ion affinity chromatography (IMAC) is a ubiquitous technique in modern recombinant production and purification. The wide range of expression vectors for the production of histidine-tagged recombinant proteins as well as the variety of stationary supports for their separation make IMAC an attractive and versatile choice for fast and reliable protein purification. It is not uncommon for IMAC purification to yield near homogenous target, protein, with purities over 95%. The small size of the histidine tag means that in many cases it can remain associated with the target protein without interference with its intended function, obviating the need for any potentially complicating tag removal steps. This chapter provides protocols for the routine purification of such histidine-tagged fusion proteins. As with any purification regime, complications with IMAC can arise. Lacking the absolute specificity of a biological ligand/ligate system such as the avidin/biotin interaction or an antibody and its cognate antigen, IMAC can sometimes display non-ideal product purity. The protocols described in this chapter provide strategies for the improvement in the purity of IMAC-purified proteins. Similarly, non-specific binding may reduce product yields and purity in some circumstances. Methodologies for enhancing the yield of the target protein are therefore provided. © Humana Press.