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

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Abstract
Epoxy supports covalently immobilize proteins following a two‐step mechanism; that is, the protein is physically adsorbed and then the covalent reaction takes place. This mechanism has been exploited to combine the selectivity of metal chelate affinity chromatography with the covalent immobilization capacity of epoxy supports. In this way, it has been possible to accomplish, 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 αβ‐heterodimeric enzyme of significant industrial interest). The selectivity of the immobilization in CES toward poly‐His‐tagged proteins was dependent to a large extent on the density and nature of the chelated metal. The highest selectivity was achieved by using low‐density chelate groups (e.g., 5 μmol/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 groups on the support significantly increased at alkaline pH values. The multipoint attachment to the CES also depended on the reaction time. The immobilization of both glutaryl acylase subunits was achieved by incubation of the enzyme derivative at pH 10 for 24 h, with the best enzyme derivative 100‐fold more stable than the soluble enzyme. By taking advantage of the selectivity 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. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 269–276, 2001.
Funding Information
  • European Community
  • Spanish CICYT
  • Resindion SRL (Mitsubishi Chemical Corp.) (B104-CT96-0005, QUI97-0506-CO3-02)