Fast and simple purification of histidine‐tagged proteins using His GraviTrap

Abstract

The histidine-tag has become the most used affinity tag to facilitate the purification and detection of recombinant proteins. Immobilized metal ion affinity chromatography (IMAC) with Ni2+ is the standard method for purifying histidine-tagged proteins. His GraviTrapTM is a new pre-packed column, filled with 1 ml Ni SepharoseTM 6 Fast Flow, for simple gravity-flow purifications of histidine-tagged proteins from clarified or unclarified samples. In this work we have used His GraviTrap to purify different histidine-tagged proteins from E.coli lysates. Clarified lysate containing approximately 40 mg of a histidine-tagged protein was applied and a recovery close to 100 % was obtained in only 40 minutes. His GraviTrap and a comparable product from another supplier were compared for purifying a histidine-tagged protein during native conditions. Results show that the total purification time was approximately five-times faster on His GraviTrap with similar purity in the eluted pools. Purification of a high molecular weight (Histidine)10-tagged protein from a clarified E.coli lysate on His GraviTrap was performed in 25 minutes. Analysis of the eluate on SDS-PAGE showed however a lot of bands. Western blot analsysis using an anti-His antibody was performed, and it was shown that the “impurities” are truncated versions of the target protein.