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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) ; 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 |
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Appears in Collections: | Journal Article |
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