( Lucia Martinez and Ario de M...)Double-Affinity Purification of GST-His Tagged Proteins Using Vivapure 8-to-96 Well Cobalt-Chelate Kit

Lucia Martinez and Ario de Marco
EMBL Protein Expression Core Facility, Meyerhofstr. 1, D-69117, Heidelberg, Germany

Introduction
Immobilized Metal ion Affinity Chromatography (IMAC) is an easy and reliable technique that allows the fast separation of His-tagged proteins from total lysates. There are several commercially available resins but all of them show some affinity for untagged proteins. Therefore, a second chromatographic step is often necessary and this implies a preventive buffer exchange step.

Affinity purification of GST-tagged proteins using glutathione sepharose usually results in lower degree of non-specific contamination, even though degradation products of the fusion proteins are often co-purified. A combined, two-step affinity purification can overcome the described drawbacks.

Here we report the purification of a 27 kDa protein fused to a double-tag (Gluthathione-S-Transferase fused to a poly-histidine tag, GST+HIS; Figure 1) using Vivapure Cobalt-loaded 8-strips. The proposed protocol does not require buffer exchange steps and includes the digestion and removal of the tags.

The first purification step exploited the GST-tag to separate the fusion protein from the bacterial proteins. The eluate was directly digested in the presence of tobacco etch virus protease (TEV protease) to remove both tags, and finally the His-tag was used to bind the double tag-moiety to the Vivapure Co-Chelate columns whereas the purified target protein was recovered in the flow-through.

Materials and methods
Cultures
A 5-ml preculture of E. coli transformed with the double-tagged fusion protein was used to inoculate 500 ml LB medium. The cells were initially incubated at 37C under agitation (180 rpm) until the OD was 0.4, then at 20C. Protein expression was induced by addition of IPTG to a final concentration of 100 M.

Pellet recovery
The cells were harvested by centrifugation (3,000 x g for 15 min). The supernatant was removed and the pellet resuspended in PBS, centrifuged again and finally frozen.

Lysate preparation
The pellet was lysed using the resuspension buffer (PBS, lysozyme 1mg/ml, DNase 1g/ml, 5 mM MgCl2), incubated for 30 min under agitation and the supernatant was recovered by centrifugation (3 min at 13,000 rpm).

Double affinity purification using Vivapure columns
The Vivapure 8-to-96 well Cobalt-Chelate kit (Vivascience AG) was used for a 2-step purification of the target protein. A scheme of the purification protocol is shown in Figure 2.

Briefly, an empty Vivaclear plus column was loaded with 150 l of glutathione sepharose resin and washed three times with 500 l of PBS before addition of the cell supernatant. The flow rate of the solutions through the column was speeded up by a centrifugation step (1 min at 6,000 rpm). The flow-through was discarded, the column was washed three times with 500 l of PBS, and the GST-tagged protein bound to the glutathione sepharose was eluted with 350 l of 20 mM Tris-HCl plus 10 mM reduced glutathione. The fusion protein was incubated for 2 h at 30C in the presence of TEV protease (1:100). The digested fraction was loaded onto a Vivapure Cobalt-Chelate column equilibrated with 500 l of 20 mM Tris-HCl. The target protein was recovered in the flow-through.

The column dead volume was considered to optimize the yield, namely the first 50 l of eluate was discarded and additional 100 l of Tris-HCl was loaded on top of the protein fraction. The fusion-tag was bound by the cobalt chelates in the column.

Results
The fractions corresponding to the lysate supernatant, glutathione-resin eluate, TEV-digested and IMAC purified were analysed by SDS-PAGE (Figure 3).

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.