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

New immobilized metal ion affinity chromatography (IMAC) matrices containin g a high concentration of metal-chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the p urification of polyhistidine (poly-His) tagged proteins. The purification o f an interesting recombinant multimeric enzyme (a thermoresistant beta -gal actosidase from Thermus sp. strain T2) has been used to check the performan ce of these new chromatographic media. IMAC supports with a high concentrat ion (and surface density) of metal chelate groups promote a rapid adsorptio n of poly-His tagged proteins during IMAC. However, these supports also fav or the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged protein s: (a) more than 30% of the natural proteins contained in crude extracts fr om 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 belong ing 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 sup ports 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 hardl y affects the rate of the one-point adsorption of poly-His tagged proteins (80% of the rate of adsorption compared to uncoated supports). Therefore, t his dextran coating of chromatographic matrices seems to allow the formatio n of strong one-point adsorptions that involve small areas of the protein a nd support surface. However, the dextran coating seems to have dramatic eff ects for the prevention of weak or strong multipoint interactions that shou ld involve a high geometrical congruence between the enzyme and the support surface. (C) 2001 Elsevier Science BN. All rights reserved.