An improved purification procedure for soluble processing alpha-glucosidase I from Saccharomyces cerevisiae overexpressing CWH41.
Processing alpha-glucosidase I, which is encoded by CWH41, regulates one of the key steps in asparagine-linked glycoprotein biosynthesis by cleaving the terminal alpha-1,2-linked glucose from Glc(3)Man(9)GlcNAc(2), the common oligosaccharide precursor. This cleavage is essential for further processing of the oligosaccharide to the complex, hybrid, and high mannose type carbohydrate structures found in eukaryotes. A method is described for the purification of the soluble form of the alpha-glucosidase I, from recombinant Saccharomyces cerevisiae overexpressing CWH41. A homogeneous enzyme preparation was obtained in higher yield than previously reported. Cultivation of recombinant S. cerevisiae in a fermenter increased the biomass 1.7 times per liter and enzyme production 2 times per liter compared to cultivation in shake flasks. Ammonium sulfate precipitation with three chromatography steps, including chromatography on an N-(5'-carboxypentyl)-1-deoxynojirimycin column, resulted in highly purified enzyme with no detectable contamination by other alpha- and beta-aryl-glycosidases. The purification procedure reproducibly yielded 40 microg of pure enzyme per gram wet biomass. Enzyme that was purified using an alternative procedure contained minor impurities and was hydrolyzed by an endogenous proteolytic activity to peptides that retained full catalytic activity. Controlled trypsin hydrolysis of the highly purified enzyme released polypeptide(s) containing the alpha-glucosidase I catalytic domain, with no loss of catalytic activity. This suggests that the catalytic domain of yeast alpha-glucosidase I is resistant to trypsin hydrolysis and remains fully functional after cleavage.
Faridmoayer
Amirreza
A
Department of Food, Nutrition, and Health, University of British Columbia, 6650 NW Marine Drive, Vancouver, BC, Canada V6T 1Z4.
Scaman
Christine H
CH
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D065089:Glycoside Hydrolase Inhibitors; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D012995:Solubility; D014357:Trypsin / Q000378:metabolism; D000520:alpha-Glucosidases / Q000096:biosynthesis / Q000302:isolation & purification*
D004791:Enzyme Inhibitors; D065089:Glycoside Hydrolase Inhibitors; D008562:Membrane Glycoproteins; D029701:Saccharomyces cerevisiae Proteins; D017485:1-Deoxynojirimycin; C065662:N-(5'-carboxypentyl)-1-deoxynojirimycin; C042059:glucosidase I; C098060:CWH41 protein, S cerevisiae; D000520:alpha-Glucosidases; D014357:Trypsin