One-Step Purification of Recombinant Proteins with the 6xHis Tag and Ni-NTA Resin

The 6xHis/Ni-NTA system is a fast and versatile tool for the affinity purification of recombinant proteins and antigenic peptides. It is based on the high-affinity binding of six consecutive histidine residues (the 6xHis tag) to immobilized nickel ions, giving a highly selective interaction that allows purification of tagged proteins or protein complexes from <1% to >95% homogeneity in just one step (1 ,2 ). The tight association between the tag and the Ni-NTA resin allows contaminants to be easily washed away under stringent conditions, yet the bound proteins can be gently eluted by competition with imidazole, or a slight reduction in pH. Moreover, because the interaction is independent of the tertiary structure of the tag, 6xHis-tagged proteins can be purified even under the strongly denaturing conditions required to solubilize inclusion bodies.