One-Step Purification, Immobilization, and Stabilization of Poly-Histidine-Tagged Enzymes Using Metal Chelate-Epoxy Supports

In this chapter, the combined use of the selectivity of metal chelate affinity chromatography with the capacity of epoxy supports to immobilize poly-His-tagged proteins via multipoint covalent attachment is shown. This has been achieved by designing tailor-made chelate-epoxy supports. In order to selectively adsorb the poly-His-tagged proteins, a very small density of Me-chelate groups was introduced in the epoxy supports. This allows for the retention of most of the epoxy groups available to multipointly react with the proteins. To reach the maximum support-enzyme reaction, after the first covalent immobilization process, the derivatives were incubated at alkaline pH conditions—where the nucleophile groups of the protein are more reactive. This protocol allows for the creation of immobilized preparations containing almost pure poly-His-tagged enzymes, even when using crude extracts, and enzymes much more stable than soluble ones.