Small epitope-linker modules for PCR-based C-terminal tagging in Saccharomyces cerevisiae

Abstract

PCR-mediated gene modification is a powerful approach to the functional analysis of genes in Saccharomyces cerevisiae. One application of this method is epitope-tagging of a gene to analyse the corresponding protein by immunological methods. However, the number of epitope tags available in a convenient format is still low, and interference with protein function by the epitope, particularly if it is large, is not uncommon. To address these limitations and broaden the utility of the method, we constructed a set of convenient template plasmids designed for PCR-based C-terminal tagging with 10 different, relatively short peptide sequences that are recognized by commercially available monoclonal antibodies. The encoded tags are FLAG, 3 x FLAG, T7, His-tag, Strep-tag II, S-tag, Myc, HSV, VSV-G and V5. The same pair of primers can be used to construct tagged alleles of a gene of interest with any of the 10 tags. In addition, a six-glycine linker sequence is inserted upstream of these tags to minimize the influence of the tag on the target protein and maximize its accessibility for antibody binding. Three marker genes, HIS3MX6, kanMX6 and hphMX4, are available for each epitope. We demonstrate the utility of the new tags for both immunoblotting and one-step affinity purification of the regulatory particle of the 26S proteasome. The set of plasmids has been deposited in the non-profit plasmid repository Addgene (http://www.addgene.org).

Figure 1

Map of the common template for the series of epitope-tagging plasmids. The positions of the six-glycine coding sequence, epitope tag coding sequence, ADH1 transcriptional terminator and selection marker between the PacI and PmeI restriction sites are shown. The DNA sequences and corresponding translated sequences between PacI and AscI that are common to all the plasmids are also shown. The general design for the forward and reverse primers is shown at the bottom

Figure 2

Application of two different epitope tags for detecting the Rpt4 proteasome subunit. (A) Growth of yeast strains expressing the indicated tagged alleles of RPT4 is indistinguishable from congenic WT cells. The strains used were YPH499 (WT), MHY4749 (RPT4–6 × GLY–T7) and MHY4913 (RPT4–6 × GLY–V5). (B) Protein extracts from OD₆₀₀ = 0.2 equivalents of the same cells were used for immunoblotting with antibodies against Rpt4, the T7 tag or the V5 tag

Figure 3

Purification of 19S regulatory particles of the 26S proteasome using a strain expressing the Rpt5–6 × Gly–FLAG protein. Yeast whole cell extracts (10 µg) from YPH499 (lane 1) and MHY4677 (lane 2) and 10 µl proteins eluted from the washed resin (lanes 3 and 4) were used for CBB staining and immunoblotting using antibodies against Rpn5 (lid), Rpt4 (base) or Pre6/α4 (20S core particle). Molecular mass standards are shown at left for the CBB-stained gel