A modified metal-ion affinity chromatography procedure for the purification of histidine-tagged recombinant proteins expressed in Drosophila S2 cells

We have developed a modified method of immobilized metal-ion affinity chrom atography (IMAC) that can be used for the purification of histidine-tagged proteins from conditioned medium containing free copper ions. Classical met hods of IMAC purification, using resins such as Ni-NTA, have proven ineffic ient for this type of purification and require multiple steps due to the in terference of divalent copper ions with the binding of His-tagged protein t o the charged resin. In contrast, this modified IMAC procedure, using chela ting Sepharose instead of Ni-NTA, enables efficient purification from coppe r-containing medium in a single step. This method appears to rely upon a pr eferential interaction of protein-copper complexes with immobilized chelati ng resin, We have utilized this method to purify active, His-tagged murine interleukin 12 from the conditioned medium of Drosophila S2 cells coexpress ing recombinant p40 and His-tagged p35 subunits and for the purification of the extracellular domain of the erythropoietin receptor. This method shoul d be applicable to the purification of a wide variety of His-tagged fusion proteins expressed in Drosophila cells and in other systems where free meta l ions are present. (C) 2000 Academic Press.