One-step purification, covalent immobilization, and additional stabilization of poly-His-tagged proteins using novel heterofunctional chelate-epoxy supports

Epoxy supports covalently immobilize proteins following a two-step mechanis m; that is, the protein is physically adsorbed and then the covalent reacti on takes place. This mechanism has been exploited to combine the selectivit y of metal chelate affinity chromatography with the covalent immobilization capacity of epoxy supports. In this way, it has been possible to accomplis h, in a simple manner, the purification, immobilization, and stabilization of a poly-His-tagged protein. To fulfill this objective we developed a new kind of multifunctional epoxy support (chelate epoxy support [CES]), which was tested using a poly-His-tagged glutaryl acylase as a model protein (an up-heterodimeric enzyme of significant industrial interest). The selectivit y of the immobilization in CES toward poly-His-tagged proteins was dependen t to a large extent on the density and nature of the chelated metal. The hi ghest selectivity was achieved by using low-density chelate groups (e.g., 5 mu mol/g) and metals with a low affinity (e.g., Co). However, the rate of covalent immobilization of the protein by its reaction with the epoxy group s on the support significantly increased at alkaline pH values. The multipo int attachment to the CES also depended on the reaction time. The immobiliz ation of both glutaryl acylase subunits was achieved by incubation of the e nzyme derivative at pH 10 for 24 h, with the best enzyme derivative 100-fol d more stable than the soluble enzyme. By taking advantage of the selectivi ty properties of the novel support, we were able to immobilize up to 30 mg of protein per gram of modified Eupergit 250 using either pure enzyme or a very crude enzyme extract. (C) 2001 John Wiley & Sons, Inc.