MULTIPURPOSE VECTORS DESIGNED FOR THE FAST GENERATION OF N-TERMINAL OR C-TERMINAL EPITOPE-TAGGED PROTEINS

In this paper are described a set of new high-copy-number yeast vector s, which are specially designed for the conditional expression of epit ope-tagged proteins in vivo. One of the major advantages of these plas mids is that they allow polymerase chain reaction-amplified open readi ng frames to be automatically fused in frame with the epitope-coding s equence, avoiding longer procedures such as site-directed mutagenesis. This heterologous construction can be realized either at the 5'-end o f 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 basi c URA3-borne pYeF1 and pYeF2 were constructed, carrying either the HIS 3 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 i mmunoprecipitation of the epitope-tagged Rna15 protein, which is invol ved in Saccharomyces cerevisiae mRNA stability.