Alejandro Vian

Affinity chromatography of polyhistidine tagged enzymes: New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions

New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal-chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant beta-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media. IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins: (a) more than 30% of the natural proteins contained in crude extracts from E. coli become adsorbed, in addition to our target recombinant protein, on these IMAC supports via multipoint weak adsorptions; (b) the multimeric poly-His tagged enzyme may become adsorbed via several poly-His tags belonging to different subunits. In this way, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole). The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions: (i) less than 2% of natural proteins contained in crude extracts are now adsorbed on these novel supports; and (ii) the target multimeric enzyme may be fully desorbed from the support using 60 mM imidazole. In spite of this dramatic reduction of multi-point interactions, this dextran coating hardly affects the rate of the one-point adsorption of poly-His tagged proteins (80% of the rate of adsorption compared to uncoated supports). Therefore, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface. However, the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface.

Stabilization of a multimeric β-galactosidase from Thermus sp. strain T2 by immobilization on novel heterofunctional epoxy supports plus aldehyde-dextran cross-linking

This work exemplifies the advantages of using a battery of new heterofunctional epoxy supports to immobilize enzymes. We have compared the performance of a standard Sepabeads‐epoxy support with other Sepabeads‐epoxy supports partially modified with boronate, iminodiacetic, metal chelates, and ethylenediamine in the immobilization of the thermostable β‐galactosidase from Thermus sp. strain T2 as a model system. Immobilization yields depended on the support, ranging from 95% using Sepabeads‐epoxy‐chelate, Sepabeads‐epoxy‐amino, or Sepabeads‐epoxy‐boronic to 5% using Sepabeads‐epoxy‐IDA. Moreover, immobilization rates were also very different when using different supports. Remarkably, the immobilized β‐galactosidase derivatives showed very improved but different stabilities after favoring multipoint covalent attachment by long‐term alkaline incubation, the enzyme immobilized on Sepabeads‐epoxy‐boronic being the most stable. This derivative had some subunits of the enzyme not covalently attached to the support (detected by SDS‐PAGE). This is a problem if the biocatalysts were to be used in food technology. The optimization of the cross‐linking with aldehyde‐dextran permitted the full stabilization of the quaternary structure of the enzyme. The optimal derivative was very active in lactose hydrolysis even at 70 °C (over 1000 IU/g), maintaining its activity after long incubation times under these conditions and with no risk of product contamination with enzyme subunits.

Overproduction of Thermus sp. Strain T2 β-Galactosidase in Escherichia coli and Preparation by Using Tailor-Made Metal Chelate Supports

ABSTRACT A novel thermostable chimeric β-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the β-galactosidase from Thermus sp. strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC). The poly-His tag fusion did not affect the activation, kinetic parameters, and stability of the β-galactosidase. Copper-iminodiacetic acid (Cu-IDA) supports enabled the most rapid adsorption of the His-tagged enzyme, favoring multisubunit interactions, but caused deleterious effects on the enzyme stability. To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-IDA or Ni-IDA supports. The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made IMAC supports.

A simple strategy for the purification of large thermophilic proteins overexpressed in mesophilic microorganisms: application to multimeric enzymes from Thermus sp. strain T2 expressed in Escherichia coli.

The heating of protein preparations of mesophilic organism (e.g., E. coli) produces the obliteration of all soluble multimeric proteins from this organism. In this way, if a multimeric enzyme from a thermophilic microorganism is expressed in these mesophilic hosts, the only large protein remaining soluble in the preparation after heating is the thermophilic enzyme. These large proteins may be then selectively adsorbed on lowly activated anionic exchangers, enabling their full purification in just these two simple steps. This strategy has been applied to the purification of an α‐galactosidase and a β‐galactosidase from Thermus sp. strain T2, both expressed in E. coli, achieving the almost full purification of both enzymes in only these two simple steps. This very simple strategy seems to be of general applicability to the purification of any thermophilic multimeric enzyme expressed in a mesophilic host.

Production of a thermoresistant alpha-galactosidase from Thermus sp. strain T2 for food processing

The gene of a thermostable alpha-galactosidase, aglA, from Thermus sp. strain T2 was sequenced, cloned and overexpressed in Escherichia coli (strain MC 2508. The purified enzyme proved to be quite thermostable, retaining high levels of activity even after incubation at 70°C. The optimal T was 65°C at pH 7. The highest activity was achieved at acid pH values, although good activity could be obtained in a broad pH range. Enzyme stability depends on the enzyme concentration at alkaline pH values, suggesting that under these conditions the subunit dissociation may be the first step in the inactivation of the enzyme. However, at pH 5 the enzyme stability becomes independent of the enzyme concentration. The enzyme also exhibited a quite broad specificity, although it shows the best activity with alpha derivatives of galactose, and was able to recognize very different substrates (alpha derivatives of mannose, xylose and maltose, and even beta galactose derivatives). There was no detectable activity against glucose derivatives.