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dc.contributor.authorHe, Xuen_US
dc.contributor.authorD. Galpin, Jasonen_US
dc.contributor.authorB. Tropak, Michaelen_US
dc.contributor.authorMahuran, Donen_US
dc.contributor.authorHaselhorst, Thomasen_US
dc.contributor.authorvon Itzstein, Marken_US
dc.contributor.authorKolarich, Danielen_US
dc.contributor.authorPacker, Nicolle H.en_US
dc.contributor.authorMiao, Yansongen_US
dc.contributor.authorJiang, Liwenen_US
dc.contributor.authorA. Grabowski, Gregoryen_US
dc.contributor.authorA. Clarke, Lorneen_US
dc.contributor.authorR. Kermode, Allisonen_US
dc.date.accessioned2017-04-24T07:56:38Z
dc.date.available2017-04-24T07:56:38Z
dc.date.issued2012en_US
dc.date.modified2014-08-28T05:07:51Z
dc.identifier.issn14602423en_US
dc.identifier.doi10.1093/glycob/cwr157en_US
dc.identifier.urihttp://hdl.handle.net/10072/42727
dc.description.abstractThere is a clear need for efficient methods to produce protein therapeutics requiring mannose-termination for therapeutic efficacy. Here we report on a unique system for production of active human lysosomal acid ߭glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45) using seeds of the Arabidopsis thaliana cgl mutant, which are deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101). Gaucher disease is a prevalent lysosomal storage disease in which affected individuals inherit mutations in the gene (GBA1) encoding GCase. A gene cassette optimized for seed expression was used to generate the human enzyme in seeds of the cgl (C5) mutant, and the recombinant GCase was mainly accumulated in the apoplast. Importantly, the enzymatic properties including kinetic parameters, IC50 of isofagomine (IFG), and thermal stability of the cgl-derived GCase were comparable to those of imiglucerase, a commercially-available recombinant human GCase used for enzyme replacement therapy in Gaucher patients. N-glycan structural analyses of recombinant cgl-GCase showed that the majority of the N-glycans (97%) were mannose-terminated. Additional purification was required to remove the ~15% of the plant-derived recombinant GCase that possessed potentially immunogenic (xylose- and/or fucose-containing) N-glycans. Uptake of cgl-derived GCase by mouse macrophages was similar to that of imiglucerase. The cgl seed system requires no addition of foreign (non-native) amino acids to the mature recombinant GCase protein, and the dry transgenic seeds represent a stable repository of the therapeutic protein. Other strategies that may completely prevent plant-like complex N-glycans are discussed, including use of a null cgl mutant.en_US
dc.description.peerreviewedYesen_US
dc.description.publicationstatusYesen_US
dc.languageEnglishen_US
dc.language.isoen_US
dc.publisherOxford University Pressen_US
dc.publisher.placeUnited Kingdomen_US
dc.relation.ispartofstudentpublicationNen_US
dc.relation.ispartofpagefrom492en_US
dc.relation.ispartofpageto503en_US
dc.relation.ispartofissue4en_US
dc.relation.ispartofjournalGlycobiologyen_US
dc.relation.ispartofvolume22en_US
dc.rights.retentionYen_US
dc.subject.fieldofresearchEnzymesen_US
dc.subject.fieldofresearchReceptors and Membrane Biologyen_US
dc.subject.fieldofresearchStructural Biology (incl. Macromolecular Modelling)en_US
dc.subject.fieldofresearchcode060107en_US
dc.subject.fieldofresearchcode060110en_US
dc.subject.fieldofresearchcode060112en_US
dc.titleProduction of active human glucocerebrosidase in seeds of Arabidopsis thaliana complex-glycan deficient (cgl) plantsen_US
dc.typeJournal articleen_US
dc.type.descriptionC1 - Peer Reviewed (HERDC)en_US
dc.type.codeC - Journal Articlesen_US
gro.date.issued2012
gro.hasfulltextNo Full Text


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