Efficient and rapid purification of recombinant human α-galactosidase A by affinity column chromatography

Abstract

The lysosomal enzyme α-galactosidase A (α-Gal A) metabolizes neutral glycosphingolipids that possess α-galactoside residues at the non-reducing terminus, and inherited defects in the activity of α-Gal A lead to Fabry disease. We describe here an efficient and rapid purification procedure for recombinant α-Gal A by sequential Concanavalin A (Con A)–Sepharose and immobilized thio-α-galactoside (thio-Gal) agarose column chromatography. Optimal elution conditions for both columns were obtained using overexpressed human α-Gal A. We recommend the use of a mixture of 0.9 M methyl α-mannoside and 0.9 M methyl α-glucoside in 0.1 M acetate buffer (pH 6.0) with 0.1 M NaCl for the maximum recovery of glycoproteins with multiple high-mannose type sugar chains from Con A column chromatography, and that the Con A column should not be reused for the purification of glycoproteins that are used for structural studies. Binding of the enzyme to the thio-Gal column requires acidic condition at pH 4.8. A galactose-containing buffer (25 mM citrate–phosphate buffer, pH 5.5, with 0.1 M galactose, and 0.1 M NaCl) was used to elute α-Gal A. This procedure is especially useful for the purification of mutant forms of α-Gal A, which are not stable under conventional purification techniques. A protocol that purifies an intracellular mutant α-Gal A (M279I) expressed in COS-7 cells within 6 h at 62% overall yield is presented.