Abstract
Immobilized metal affinity chromatography (IMAC) purification of secreted histidine-tagged proteins in eukaryotic cell culture supernatants is often problematic. Incompatibility with the cell culture media appears as stripping of the immobilized metal ions required for binding of target proteins. The stripping effect is aggravated by low target protein concentrations and hence large sample volumes.
In this study, purifications of histidine-tagged proteins were performed using two novel Sepharose™ based IMAC media; Ni Sepharose excel and magnetic His Mag Sepharose excel. Both media have a new type of chelating ligand with exceptionally strong binding of nickel ions. Data showing successful purification of histidine-tagged proteins from CHO cell culture supernatants will be presented. In comparison, conventional IMAC medium showed no recovery of target protein.
Furthermore, the characteristics of new media enabled purification of target protein from insect cell culture supernatants. The purification was easily scaled up from 20 μl His Mag Sepharose excel beads to 1 mL pre-packed columns.