Column Purification of Histidine-Tagged Proteins from Unclarified Samples

The histidine-tag is today the most commonly used tag for recombinant proteins. It is widely used to facilitate purification of recombinant expressed proteins, but can also be useful for detection purposes. For immobilized metal-ion affinity chromatography (IMAC) the most used metal ion is nickel (Ni2+). However, also the cobalt ion (Co2+) has been shown useful in obtaining high purity with good yields of, especially low expressed, histidine-tagged target proteins in IMAC applications. Pre-packed HiTrapTM IMAC crude column is a format which facilitates scalable (1-ml and 5-ml columns), fast and convenient protein purifications of histidine-tagged proteins. Un-clarified samples can be loaded directly to the column. The process time is decreased which helps to ensure high concentrated pure and intact target proteins with minimal degradation. Several applications were performed to verify the performance of pre-packed HiTrap IMAC crude column. Two histidine-tagged proteins (produced in E. coli and P. pastoris expression systems) were purified ending up in 90% purity and yields of 90%. A fully automated (AKTAxpressTM) purification of a histidine-tagged kinase (1 mg/g E. coli cells) has been performed. The purification was done by IMAC and gel filtration. According to SDS-PAGE analysis the obtained purity of the kinase was above 90% and the amount purified protein was 3 mg. Further, four purification cycles, without cleaning and Co2+-recharging between the runs; show that the HiTrap IMAC crude columns can be used several times without losing performance. Purity and yield was unchanged (within + 5%). Pre-packed HiTrap TALON® crude columns are very convenient for rapid and easy purification of histidine-tagged proteins, also when using Co2+-charged resins, resulting in high purity and yield of the target protein(s). In addition the column can be reused several times (with no need for regeneration between runs) without loss of performance.