Please use this identifier to cite or link to this item: https://hdl.handle.net/1959.11/20512
Title: Production and crystallization of processing α-glucosidase I: 'Pichia pastoris' expression and a two-step purification toward structural determination
Contributor(s): Barker, Megan K (author); Wilkinson, Brendan  (author)orcid ; Faridmoayer, Amirreza (author); Scaman, Christine H (author); Fairbanks, Antony J (author); Rose, David R (author)
Publication Date: 2011
DOI: 10.1016/j.pep.2011.05.015
Handle Link: https://hdl.handle.net/1959.11/20512
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 4.2 mg Cwht1p per liter of growth culture. To test catalytic activity, we developed a modified synthesis of a tetrasaccharide substrate, Glc₃ManOMe. Cwht1p with Glc₃ManOMe shows a Km 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.
Publication Type: Journal Article
Source of Publication: Protein Expression and Purification, 79(1), p. 96-101
Publisher: Academic Press
Place of Publication: United States of America
ISSN: 1096-0279
1046-5928
Fields of Research (FoR) 2008: 060112 Structural Biology (incl. Macromolecular Modelling)
030403 Characterisation of Biological Macromolecules
030406 Proteins and Peptides
Fields of Research (FoR) 2020: 340407 Proteins and peptides
340403 Characterisation of biological macromolecules
310112 Structural biology (incl. macromolecular modelling)
Socio-Economic Objective (SEO) 2008: 970103 Expanding Knowledge in the Chemical Sciences
970106 Expanding Knowledge in the Biological Sciences
Socio-Economic Objective (SEO) 2020: 280102 Expanding knowledge in the biological sciences
280105 Expanding knowledge in the chemical sciences
Peer Reviewed: Yes
HERDC Category Description: C1 Refereed Article in a Scholarly Journal
Appears in Collections:Journal Article

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