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Campo DC | Valor | Lengua/Idioma |
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dc.contributor.author | Bastida, Agatha | - |
dc.contributor.author | Fernández-Mayoralas, Alfonso | - |
dc.contributor.author | Gómez Arrayás, Ramón | - |
dc.contributor.author | Iradier, Fátima | - |
dc.contributor.author | Carretero, Juan Carlos | - |
dc.contributor.author | García-Junceda, Eduardo | - |
dc.date.accessioned | 2009-03-16T15:34:21Z | - |
dc.date.available | 2009-03-16T15:34:21Z | - |
dc.date.issued | 2001 | - |
dc.identifier.citation | Chemistry 2001, 7, 2390-2397 | en_US |
dc.identifier.issn | 0947-6539 | - |
dc.identifier.uri | http://hdl.handle.net/10261/11652 | - |
dc.description.abstract | An efficient heterologous expression system for overproduction of the enzyme alpha 1,6-Fucosyltransferase (alpha 1,6-FucT) from Rhizobium sp. has been developed. The gene codifying for the alpha 1,6-FucT was amplyfied by PCR using specific primers. After purification, the gene was cloned in the plasmid pKK223-3. The resulting plasmid, pKK1,6FucT, was transformed into the E. coli strain XL1-Blue MRF’. The protein was expressed both as inclusion bodies and in soluble form. Changing the induction time a five-fold increase of enzyme expressed in soluble form was obtained. In this way 5 units of enzyme alpha 1,6-FucT can be obtained per liter of culture. A crude preparation of the recombinant enzyme was used for the synthesis of the branched trisaccharide beta-D-GlcNAc-(1-4)-[alpha-L-Fuc-(1-6)]-D-GlcNAc (3), from chitobiose (2) and GDP-Fucose (1). After purification, the trisaccharide 3 was obtained in a 84% overall yield. In order to know the structural requirements for acceptors, the specificity of the enzyme was studied towards mono-, di- and trisaccharides structuraly related to chitobiose. The enzyme is able to use, among others, the disaccharide N-acetyl lactosamine (4) as a good substrate and the monosaccharide GlcNAc (8) as a weak acceptor. Finally, several racemic polyhydroxylated indolizidines have been tested as potencial inhibitors of the enzyme. The indolizidine 21 was the best inhibitor with an IC50 of 4.5 x 10-5 M and, interestingly, is the one that could better mimic the structural features of the fucose moiety in the presumed transition state. | en_US |
dc.description.sponsorship | This work was supported by the Spanish DGES (Grant PB96-0828) and Comunidad de Madrid (Grants 07B/0027/1999 and 07B/0028/1999). | en_US |
dc.format.extent | 237169 bytes | - |
dc.format.mimetype | application/pdf | - |
dc.language.iso | eng | en_US |
dc.publisher | Wiley-VCH | en_US |
dc.rights | openAccess | en_US |
dc.subject | enzyme alpha 1,6-Fucosyltransferase | en_US |
dc.subject | Rhizobium sp | en_US |
dc.subject | Trisaccharide beta-D-GlcNAc(1-4)-[alpha-L-Fuc-(1-6)]-D-GlcNAc | en_US |
dc.subject | Polyhydroxylated Indolizidines | en_US |
dc.title | Heterologous Over-expression of alpha 1,6-Fucosyltransferase from Rhizobium sp. Application to the Synthesis of the Trisaccharide beta-D-GlcNAc(1-4)-[alpha-L-Fuc-(1-6)]-D-GlcNAc, Study of the Acceptor Specificity, and Evaluation of Polyhydroxylated Indolizidines as Inhibitors. | en_US |
dc.type | artículo | en_US |
dc.identifier.doi | 10.1002/1521-3765(20010601)7:11<2390::AID-CHEM23900>3.0.CO;2-0 | - |
dc.description.peerreviewed | Peer reviewed | en_US |
dc.relation.publisherversion | http://dx.doi.org/10.1002/1521-3765(20010601)7:11<2390::AID-CHEM23900>3.0.CO;2-0 | en_US |
dc.identifier.e-issn | 1521-3765 | - |
dc.type.coar | http://purl.org/coar/resource_type/c_6501 | es_ES |
item.openairetype | artículo | - |
item.grantfulltext | open | - |
item.cerifentitytype | Publications | - |
item.openairecristype | http://purl.org/coar/resource_type/c_18cf | - |
item.fulltext | With Fulltext | - |
item.languageiso639-1 | en | - |
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Heterologous Over-expression of alpha 1,6-Fucosyltransferase.pdf | 231,61 kB | Adobe PDF | Visualizar/Abrir |
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