One Step Purification Immobilization and Stabilization of Poly HistidineTagged Enzymes Using Metal Chelate Epoxy Supports

Cesar Mateo, Benevides C. C. Pessela, Valeria Grazu, Rodrigo Torres, Fernando Lopez-Gallego, Jose M. Guisan, and Roberto Fernandez-Lafuente

Summary

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.

Key Words: Tailor-made IMAC supports; heterofunctional supports; selective adsorption of enzymes; oriented immobilization; enzyme stabilization by multipoint covalent attachment.

1. Introduction

The use of pure enzyme to prepare immobilized enzyme derivatives may be very relevant in biocatalysis. This may allow for improvement of the volumetric activity of the support (the only immobilized enzyme would be our target one) and avoids undesired side reactions that may be catalyzed by contaminant enzymes. However, purification of proteins is a process that may complicate the implementation of a biocatalyst because it is time consuming, high in cost, and may have

Epoxy Ida Coupling

Fig. 1. Preparation of metal chelate-epoxy supports. Epoxy supports are incubated in the presence of iminodiacetic acid to introduce a few IDA groups in the support. The IDA supports are incubated with metal salts to obtain metal chelate-epoxy supports. Remained epoxy groups are blocked incubated them in presence of mercaptoethanol.

Fig. 1. Preparation of metal chelate-epoxy supports. Epoxy supports are incubated in the presence of iminodiacetic acid to introduce a few IDA groups in the support. The IDA supports are incubated with metal salts to obtain metal chelate-epoxy supports. Remained epoxy groups are blocked incubated them in presence of mercaptoethanol.

only partial yield (even just by enzyme inactivation). One suitable solution would be to join the purification and immobilization steps by designing a highly selective immobilization process.

Epoxy supports have been described as very suitable for the immobilization and stabilization of proteins via multipoint covalent attachment (1-10).

The mechanism of enzyme immobilization in epoxy supports (11-14) provides new opportunities for coupling immobilization to purification. It has been noted that the previous adsorption of protein on the epoxy support is necessary to achieve a significant covalent immobilization of the proteins because of the extremely low reactivity of the epoxy supports with soluble proteins (11-14). Using this premise, the use of multifunctional epoxy supports to immobilize proteins has recently been reported (14): This second generation of epoxy supports has different moieties that are able to physically adsorb proteins via different structural features, as well as a dense layer of epoxy groups that are able to covalently react with the enzyme. One type of multifunctional support that may be easily produced is the metal chelate-epoxy supports (CES; see Fig. 1) (15).

These supports may combine the good properties of epoxy supports for enzyme immobilization-stabilization with the enormous possibilities of immobilized metal chelate affinity chromatography (IMAC chromatography) to purify poly-His-tagged proteins (see Fig. 2). In this case, desorption of the contaminant adsorbed proteins is not possible, so higher selectivity than conventional IMAC chromatography will be required.

To achieve this selectivity,we have taken into account that natural proteins and poly-His tagged protein become adsorbed on IMAC supports following different mechanisms. The first requires interaction between different groups on the protein surface with several metal-chelate groups (see Fig. 3) (5), whereas poly-His-tagged proteins may be adsorbed by the interaction of the His tag with just one residue in the support (5) (see Fig. 3). This has been achieved by using low-superficial density of metal chelate in the support and metals that yield low-adsorption strength (e.g., Co2+; see Fig. 3).

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