Efficient and rapid purification of recombinant human alpha-galactosidase A by affinity column chromatography.
The lysosomal enzyme alpha-galactosidase A (alpha-Gal A) metabolizes neutral glycosphingolipids that possess alpha-galactoside residues at the non-reducing terminus, and inherited defects in the activity of alpha-Gal A lead to Fabry disease. We describe here an efficient and rapid purification procedure for recombinant alpha-Gal A by sequential Concanavalin A (Con A)-Sepharose and immobilized thio-alpha-galactoside (thio-Gal) agarose column chromatography. Optimal elution conditions for both columns were obtained using overexpressed human alpha-Gal A. We recommend the use of a mixture of 0.9 M methyl alpha-mannoside and 0.9 M methyl alpha-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 alpha-Gal A. This procedure is especially useful for the purification of mutant forms of alpha-Gal A, which are not stable under conventional purification techniques. A protocol that purifies an intracellular mutant alpha-Gal A (M279I) expressed in COS-7 cells within 6h at 62% overall yield is presented.
Yasuda
Kayo
K
Department of Human Genetics, Mount Sinai School of Medicine, Box 1498, Fifth Avenue at 100th Street, New York, NY 10029, USA.
Chang
Hui-Hwa
HH
Wu
Hui-Li
HL
Ishii
Satoshi
S
Fan
Jian-Qiang
JQ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods*; D019556:COS Cells; D002460:Cell Line; D002845:Chromatography; D003470:Culture Media / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000737:chemistry; D006028:Glycosphingolipids / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D008247:Lysosomes / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry*; D014162:Transfection; D014462:Ultrafiltration; D000519:alpha-Galactosidase / Q000737:chemistry* / Q000378:metabolism
D003470:Culture Media; D006028:Glycosphingolipids; D011994:Recombinant Proteins; D000519:alpha-Galactosidase; D005690:Galactose