| Abstract |
Epitope tagging is a powerful strategy for analyzing the functions of targeted proteins. The use of this strategy has become more convenient with the development of the epitope switch, which is another type of epitope tagging designed to convert the previously tagged epitopes on the chromosome to other epitopes of interest. Various modules for C-terminal epitope switching have been developed and amplified using the one-step polymerase chain reaction (PCR) method before transformation. However, PCR amplification occasionally generates mutations that affect the fidelity of epitope switching. Here, we constructed several plasmids to isolate modules for epitope switching through digestion by restriction enzymes. The isolated modules contained DNA sequences for homologous recombination, various epitopes (13×Myc, 6×HA, GFP, Venus, YFP, mCherry, and CFP), and a transformation marker (Candida glabrata LEU2). The restriction enzyme-digested plasmids were used to directly transform the cells for epitope switching. We demonstrate the efficient and accurate switching of the MX6 module-based C-terminal tandem affinity purification tags to each aforementioned epitope. We believe that our plasmids can serve as powerful tools for the functional analysis of yeast proteins.
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