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Epitope-Tagging Vectors for Functional Analysis in Yeast

re luciferase activity. In both cases, strong luciferase activity was detected when the promoters were induced (Figure 2, lanes 4 and 6). Only very low levels of luciferase activity were detected when the promoters were repressed (Figure 2, lanes 3 and 5).

Figure 2

This assay demonstrated the following: The carbon source in the media strictly regulated the GAL1 and GAL10 promoters in the pESC vectors, the galactose-induced levels achieved greater than a 1000-fold increase in luciferase activity, and the induced levels were comparable for both promotors.

Epitope Tagging of Luciferase Using pESC Vectors

Epitope tagging is a convenient technique in which a known peptide epitope that is recognized by an antibody is fused to the target protein of interest. With this technique, a tag-specific antibody can be used to detect the fusion protein within a cell, eliminating the need to make specific antibodies to each new protein under study.9

To test the FLAG and c-myc epitope tags in the pESC vectors, we cloned the luciferase gene downstream of the GAL promoters such that the gene is fused with c-myc at the C-terminus or with FLAG at the N-terminus. For C-terminal tagging with c-myc, the firefly luciferase gene was PCR amplified and cloned into the BamH I/Sal I sites of the pESC vectors, resulting in pESC-XXX-LucMC constructs. (XXX represents a plasmid backbone with a selectable marker, such as LEU2, HIS3, TRP1, or URA3.) For N-terminal tagging with FLAG, the luciferase gene was cloned in the Bgl II site of the pESC vectors, resulting in pESC-XXX-LucFN constructs. The constructs were transformed into S. cerevisiae, strain YPH499, and the transformants were selected on synthetic dropout plates specific for the particular vector used. For example, transformants with pESC-URA-L
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