The recombinant fusion protein was the major protein detectable in the supernatant after lysate centrifugation (lane S). The first purification step was performed exploiting the affinity of GST for glutathione sepharose, and enabled the recovery of a highly purified fusion protein after elution in Tris buffer (lane E). Such buffer is compatible with TEV digestion, therefore the eluate could be directly incubated in the presence of the protease. After proteolytic removal of the tags, two bands clearly appeared on the SDS-gel, corresponding to the GST-HIS-TEV recognition site moiety and the target protein (lane D). At this point, the target protein was separated from the moiety containing the His-tag by IMAC. After loading the digested fraction onto a Vivapure Cobalt-Chelate column, the untagged target protein was recovered in the flow-through (lane P) whereas the His-tagged GST remained bound to the column. The final protein yield was in the range of 350 g for each column. Few minutes were sufficient to perform each of the column purification steps.

Conclusions
Our results show that a protein construct expressed as a GST-HIS fusion with a protease recognition site as a linker is suitable for being quickly purified using two affinity columns. This protocol provides the possibility to eliminate contaminant native proteins and to separate the target protein from the fusion partners using the Vivapure 8-to-96 well Cobalt-Chelate kit. Such a system can be easily automated and the yields, in the range of 0.5 mg/column, are largely sufficient for most lab applications such as pull-down or activity tests. The modularity of the column system enables to scale up the yields to several mg without necessity to modify the basic protocol to reach amounts sufficient for robot-driven crystallography screening.

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