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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 w
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