In vitro assay of the glycosyltransferase activity of a heterologously expressed plant protein

Show simple item record Ade, CP en Bemm, F en Dickson, James en Walter, C en Harris, Philip en 2015-11-03T22:33:14Z en 2014-11-05 en
dc.identifier.citation Bio-protocol, 2014, 4 (21) en
dc.identifier.uri en
dc.description.abstract Glycosyltransferases are carbohydrate active enzymes containing catalytic modules involved in catalysing the biosynthesis of glycosidic bonds in oligo- and polysaccharides and glycoconjugates. One of the most comprehensive collections of Carbohydrate Active enZYmes is the CAZy database ( comprising 120,000 glycosyltransferases allocated to 96 families based mainly on sequence homologies of their conserved and catalytically active domains (Cantarel et al., 2009). Interestingly, the glycosyltransferase activities of only about 1.6% of these proteins have been experimentally characterized (Lombard et al., 2014). In recent years, membrane-bound glycosyltransferases of a number of families have been shown to play a key role in the biosynthesis of plant cell-wall polysaccharides (Doblin et al., 2010; Scheller and Ulvskov, 2010; Driouich et al., 2012). They catalyze the transfer of glycosyl residues from donor nucleotide sugars to acceptors, forming the glycosidic bonds between adjacent glycosyl residues. Family 34 contains glycosyltransferases that have been shown to be involved in the biosynthesis of xyloglucans and transfer xylosyl residues to (1→4)-β-glucan chains (Keegstra and Cavalier, 2011). Our previous work suggests that Pinus radiata protein PrGT34B is a xyloglucan (1→6)-α-xylosyltransferase (Ade et al., 2014). Here, we describe a procedure for determining the xylosyltransferase activity of PrGT34B in vitro. We measured the transfer of xylose from the donor substrate UDP-xylose to different cello-oligosaccharide acceptor substrates under controlled reaction conditions. The assays include quantification of radioactively labeled reaction products and their identification by mass spectrometry. We also describe the purification, identification and quantification of the heterologously expressed recombinant protein PrGT34B in preparation for its use in the assays. This procedure may be applied to a wide range of glycosyltransferases in many different plant species. en
dc.language English en
dc.publisher Bio-Protocol en
dc.relation.ispartofseries Bio-protocol en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. Details obtained from en
dc.rights.uri en
dc.title In vitro assay of the glycosyltransferase activity of a heterologously expressed plant protein en
dc.type Journal Article en
pubs.issue 21 en
pubs.volume 4 en
dc.rights.holder Copyright: Bio-Protocol en en
pubs.publication-status Published en
dc.rights.accessrights en
pubs.subtype Article en
pubs.elements-id 502243 en Science en Biological Sciences en
dc.identifier.eissn 2331-8325 en
pubs.number e1285 en
pubs.record-created-at-source-date 2015-10-21 en

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