Multipurpose vectors designed for the fast generation of N- or C-terminal epitope-tagged proteins.
In this paper are described a set of new high-copy-number yeast vectors, which are specially designed for the conditional expression of epitope-tagged proteins in vivo. One of the major advantages of these plasmids is that they allow polymerase chain reaction-amplified open reading frames to be automatically fused in frame with the epitope-coding sequence, avoiding longer procedures such as site-directed mutagenesis. This heterologous construction can be realized either at the 5'-end of the coding sequence, in the pYeF1 vector, or at its 3'-end, in pYeF2, generating N- or C-terminal tagged proteins, respectively. Moreover, to increase the usefulness of the method, derivatives of the two basic URA3-borne pYeF1 and pYeF2 were constructed, carrying either the HIS3 or TRP1 gene as a marker of selection. These vectors could be of use for the purpose of functional analysis of the newly discovered genes resulting from the systematic sequencing of the yeast genome. Here, we present results showing the functional expression and the efficient immunoprecipitation of the epitope-tagged Rna15 protein, which is involved in Saccharomyces cerevisiae mRNA stability.
Cullin
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Centre de Génétique Moléculaire du C.N.R.S., Laboratoire Propre Associé à l'Université Pierre-et-Marie-Curie, Gif-sur-Yvette, France.
Minvielle-Sebastia
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D000595:Amino Acid Sequence; D001483:Base Sequence; D000939:Epitopes / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D005813:Genes, Synthetic; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016601:RNA-Binding Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors
D000939:Epitopes; D005656:Fungal Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; C512635:RNA15 protein, S cerevisiae