Overexpression, purification, and partial characterization of Saccharomyces cerevisiae processing alpha glucosidase I.
The gene encoding yeast processing alpha glucosidase I, CWH41, was overexpressed in Saccharomyces cerevisiae AH22, resulting in a 28-fold increase in expression of the soluble form of the enzyme. The soluble enzyme results from proteolytic cleavage between residues Ala 24 and Thr 25 of the transmembrane sequence of the membrane-bound form of the enzyme. This cleavage could be partially inhibited by addition of leupeptin and pepstatin during the enzyme isolation. The enzyme was purified to a final specific activity of 8550 U/mg protein using a combination of ammonium sulfate precipitation, anion exchange, concanavalin A, and gel filtration chromatography. The soluble form of the enzyme is a monomer with a molecular weight of 98 kDa by SDS-PAGE, and 89 kDa by gel filtration. The molecular weight decreased by approximately 5 kDa after treatment with N-glycosidase F, indicating that it is a glycoprotein. Soluble glucosidase I was sensitive to diethyl pyrocarbonate and not affected by N-ethylmaleimide, suggesting that mechanistically it is more similar to the plant than the mammalian form of the enzyme.
Dhanawansa
Ranjani
R
Food, Nutrition and Health, University of British Columbia, 6650 NW Marine Drive, Vancouver, BC, V6T 1Z4, Canada.
Faridmoayer
Amirreza
A
van der Merwe
George
G
Li
Ying X
YX
Scaman
Christine H
CH
D001483:Base Sequence; D017931:DNA Primers; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; C042059:glucosidase I; D000520:alpha-Glucosidases; D005033:Ethylmaleimide