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dc.contributor.author | Drake, Rocío | - |
dc.contributor.author | Serrano, Aurelio | - |
dc.contributor.author | Pérez-Castiñeira, J. R. | - |
dc.date.accessioned | 2019-03-28T07:44:10Z | - |
dc.date.available | 2019-03-28T07:44:10Z | - |
dc.date.issued | 2010 | - |
dc.identifier | issn: 0264-6021 | - |
dc.identifier.citation | Biochemical Journal 426: 147- 157 (2010) | - |
dc.identifier.uri | http://hdl.handle.net/10261/178709 | - |
dc.description.abstract | Expression of heterologous multispanning membrane proteins in Saccharomyces cerevisiae is a difficult task. Quite often, the use of multicopy plasmids where the foreign gene is under the control of a strong promoter does not guarantee efficient production of the corresponding protein. Here, we show that the expression level and/or subcellular localization in S. cerevisiae of an heterologous type of multispanning membrane protein, the H+-translocating inorganic pyrophosphatase (H+-PPase), can be changed by fusing it with various suitable Nterminal signal sequences. Chimeric proteins were constructed by adding the putative Nterminal extra domain of Trypanosoma cruzi H+-PPase or the bona fide signal sequence of S. cerevisiae invertase Suc2p to H+-PPase polypeptides of different organisms (from bacteria to plants) and expressed in a yeast conditional mutant deficient in its cytosolic PPi hydrolysis activity when grown on glucose. Chimeric constructs not only substantially enhanced H+- PPase expression levels in transformed mutant cells but also allowed functional complementation in those cases in which native H+-PPase failed to accomplish it. Activity assays and Western blot analyses further demonstrated the occurrence of most H+-PPase in internal membrane fractions of these cells. The addition of N-terminal signal sequences to the vacuolar H+-PPase AVP1 from the plant Arabidopsis thaliana -a protein efficiently expressed in yeast in its natural form- alters the subcellular distribution of the chimeras suggesting further progression along the secretory sorting pathways, as shown by density gradient ultracentrifugation and in vivo fluorescence microscopy of the corresponding GFP-H+-PPase fusion proteins. | - |
dc.publisher | Portland Press | - |
dc.relation.isversionof | Postprint | - |
dc.rights | openAccess | - |
dc.subject | N-terminal signal sequences | - |
dc.subject | Heterologous expression | - |
dc.subject | Saccharomyces cerevisiae | - |
dc.subject | Green fluorescent protein | - |
dc.subject | Proton-translocating inorganic pyrophosphatase | - |
dc.subject | Chimeric proteins | - |
dc.title | N-terminal chimaeras with signal sequences enhance the functional expression and alter the subcellular localization of heterologous membrane-bound inorganic pyrophosphatases in yeast | - |
dc.type | artículo | - |
dc.date.updated | 2019-03-28T07:44:10Z | - |
dc.description.version | Peer Reviewed | - |
dc.language.rfc3066 | eng | - |
dc.relation.csic | Sí | - |
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 | - |
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