Abstract
The present purification protocol applies to target proteins that are fused to a double tag, such as NusA–His6, through a linker that includes a protease-recognition sequence. It involves two steps of immobilized metal ion affinity chromatography (IMAC). NusA stabilizes the passenger protein during translation, whereas the His-tag enables affinity purification of the fusion. The eluate resulting from the first IMAC is buffer-exchanged to remove the imidazole and to achieve optimal conditions for the enzymatic cleavage performed by a His-tagged recombinant protease. The digested sample is loaded directly for a second IMAC step and the target protein is selectively recovered in the flow-through. The resin binds residual non-digested fusion protein, double-tagged moiety, protease and any contaminant that bound the affinity resin and was eluted from the first IMAC. The purity of the target protein usually makes a further purification step unnecessary for most of the lab applications. It takes less than 5 hours to purify the protein from a 5 g pellet.
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de Marco, A. Two-step metal affinity purification of double-tagged (NusA–His6) fusion proteins. Nat Protoc 1, 1538–1543 (2006). https://doi.org/10.1038/nprot.2006.289
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DOI: https://doi.org/10.1038/nprot.2006.289
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