Figure 6.17 Schematic representation of the basic principles of metal chelate affinity chromatography. Certain proteins are retained on the column via the formation of coordinate bonds with the immobilized metal ion (a). The actual structure of the most commonly used metal chelator, iminodiacetic acid, is presented in (b)

are attracted to the metal ions, forming the weak coordinate bonds. Elution of bound proteins is undertaken by lowering the buffer pH (this causes protonation of the histidine residues, which are then unable to coordinate with the metal ion). Alternatively, a strong competitor complexing agent (e.g. the chelating agent ethylenediaminetetraacetic acid (EDTA)) can be added to the elu-tion buffer.

Metal chelate affinity chromatography finds most prominent application in the affinity purification of recombinant proteins to which a histidine tag has been attached (described later). As protein binding occurs via the histidine residues, this technique is no more inherently useful for the purification of metalloproteins than for the purification of non-metalloproteins (a common misconception, given its name).

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