Epitope-Tagging Vectors for Functional Analysis in Yeast ( es that can be specifically ...)

Epitope-Tagging Vectors for Functional Analysis in Yeast

es that can be specifically recognized by monoclonal antibodies. A sequence for the FLAG epitope (DYKDDDDK⁷) is located in the MCS downstream of the GAL1 promoter; a sequence for the c-myc epitope (EQKLISEEDL⁸) is located in the MCS downstream of the GAL10 promoter (Figure 1). The gene of interest can be inserted in front of the epitope sequence to generate C-terminal tagging or be inserted after the epitope sequence to generate N-terminal tagging.

Comparing GAL1 and GAL10 Promoter Strengths

We tested the pESC vectors by inserting the firefly luciferase gene into the MCS downstream of the GAL1 or GAL10 promoter. To compare the promoter activity under repressed and induced conditions, the luciferase gene was individually cloned in the BamH I site or the Not I site of pESC-TRP, so that the gene is under the control of the GAL1 promoter or the GAL10 promoter, respectively. Correct clones were identified by PCR using primers flanking the MCS downstream of the promoters.

The resulting constructs were designated pESC-TRP-Luc (GAL1) and pESC-TRP-Luc (GAL10). Note that in the two constructs, the luciferase gene was not fused to the epitope tags. These two constructs were transformed separately into S. cerevisiae, strain YPH499, and transformants were selected on media lacking the amino acid tryptophan (SD-TRP plates). The recovered transformants were inoculated into selective media with glucose or galactose as the carbon source (i.e., SD-TRP and SG-TRP, respectively). After incubating for 16 hours, at 30C, yeast cells were collected and lysed by vortexing with acid-washed glass beads. The cleared cell lysate was used to measu
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