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Epitope-Tagging Vectors for Functional Analysis in Yeast

ucMC or pESC-URA-LucFN were selected on SD-URA plates.

Figure 3

The transformants were grown in selective media with glucose or galactose as the carbon source, and the cleared cell lysate was assayed for luciferase activity. As shown in Figure 3, all eight constructs expressed active luciferase. When pESC-TRP-LucFN or pESC-TRP-LucMC was used, under induced conditions, the activity of the epitope-tagged luciferase showed a level similar to that of the untagged luciferase (Figure 2). The data indicate that the epitope-tagged luciferase retains its biological activity, and the tags do not interfere with the enzyme activity. We consistently observed that the luciferase activities derived from the pESC-URA vector are approximately two- to three-fold less, compared to those derived from other pESC vectors (Figure 3). Lower levels of luciferase expression were most likely due to the URA3 selectable marker contained in the vector. While the HIS3, LEU2, and TRP1 gene products are involved in amino acid biosynthesis, the URA3 gene product is involved in uracil biosynthesis. Marker gene activity has been shown to influence vector maintenance and levels of gene expression in yeast.10 To detect the epitope-tagged luciferase, we subjected the yeast lysates to Western blot analysis using antibodies specific to the epitope tag. A 64-kDa band, corresponding to the expected size of the epitope-tagged luciferase protein, was detected in samples derived from cells grown in galactose media (Figure 4, even lanes) but not in samples from cells grown in glucose media (Figure 4, odd lanes).

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