title: Production and crystallization of processing α-glucosidase I: Pichia pastoris expression and a two-step purification toward structural determination.
abstract: Eukaryotic N-glycoprotein processing in the endoplasmic reticulum begins with the catalytic action of processing α-glucosidase I (αGlu). αGlu trims the terminal glucose from nascent glycoproteins in an inverting-mechanism glycoside hydrolysis reaction. αGlu has been studied in terms of kinetic parameters and potential key residues; however, the active site is unknown. A structural model would yield important insights into the reaction mechanism. A model would also be useful in developing specific therapeutics, as αGlu is a viable drug target against viruses with glycosylated envelope proteins. However, due to lack of a high-yielding overexpression and purification scheme, no eukaryotic structural model of αGlu has been determined. To address this issue, we overexpressed the Saccharomyces cerevisiae soluble αGlu, Cwht1p, in the host Pichia pastoris. It was purified in a simple two-step protocol, with a final yield of 4.2mg Cwht1p per liter of growth culture. To test catalytic activity, we developed a modified synthesis of a tetrasaccharide substrate, Glc(3)ManOMe. Cwht1p with Glc(3)ManOMe shows a K(m) of 1.26 mM. Cwht1p crystals were grown and subjected to X-ray irradiation, giving a complete diffraction dataset to 2.04 Å resolution. Work is ongoing to obtain phases so that we may further understand this fundamental member of the N-glycosylation pathway through the discovery of its molecular structure.
authors.affiliation: Department of Medical Biophysics, University of Toronto, Toronto, Canada.
authors.forename: Megan K
authors.initials: MK
authors.lastname: Barker
authors.forename: Brendan L
authors.initials: BL
authors.lastname: Wilkinson
authors.forename: Amirreza
authors.initials: A
authors.lastname: Faridmoayer
authors.forename: Christine H
authors.initials: CH
authors.lastname: Scaman
authors.forename: Antony J
authors.initials: AJ
authors.lastname: Fairbanks
authors.forename: David R
authors.initials: DR
authors.lastname: Rose
mesh_terms: D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D015870:Gene Expression; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: C042059:glucosidase I; D000520:alpha-Glucosidases