PCR-Mediated Epitope Tagging of Genes in Yeast

Epitope tagging of genes is a powerful technique facilitating assays for gene function, determination of subcellular distribution of proteins, afﬁ nity puriﬁ cation, study of protein interaction with other proteins, DNA or RNA, and any other antibody-based approach in the absence of protein-speciﬁ c antibodies. Here, we describe a one-step PCR-based strategy for insertion of epitope tags at the chromosomal locus. This method takes advantage of efﬁ cient homologous recombination in yeast. PCR ampliﬁ ed tags are directed to desired chromosomal loci with the help of primer-encoded ﬂ anking homologous sequences enabling selective epitope tagging of genes of interest.


Introduction
Saccharomyces cerevisiae is one of the most extensively studied eukaryotic model organisms in molecular and cellular biology. One of the characteristics that facilitate its use in rigorous genetic and biochemical analyses is highly active homologous recombination. A gene of interest can be effi ciently and accurately replaced or tagged by transformation with a heterologous DNA fragment bearing as little as 40 nucleotides homology at its ends to the target sequence [ 1 , 2 ].
Epitope tagging of open reading frames in yeast is a routine molecular biology procedure and is a valuable tool for studying proteins. The strategy requires a pair of primers that contain within their 5′ region, around 40-nucleotides of homology to the genomic region of interest and around 20-nucleotide homology at their 3′end to the tag cassette (Fig. 1 ). A large array of cassette plasmids with a wide variety of tags (Table 1 ) in combination with different selection markers is now readily available for use in yeast [ 3 , 4 ]. These plasmids mostly comprise of the same set of linker regions fl anking the tag cassette such that a small set of primers can be used for an assortment of genome manipulations [ 3 , 5 ]. Epitope tags are added either at the carboxyl (C) or amino (N) terminus of a protein. N-terminal tags typically rely on the use of heterologous promoters, though two-step strategies exist to tag genes at their N-termini under the control of their natural promoters [ 6 ].
Tag cassettes are PCR-amplifi ed using these homology-bearing primers, and the PCR products are transformed directly into yeast. Transformants are selected based on the auxotrophic or antibioticresistance marker linked to the epitope tag. Successful epitope tagging is confi rmed by immunoblotting to detect the protein of interest fused to the epitope tag.

Yeast Transformation
All steps at room temperature unless otherwise stated.
1. Grow a starter culture of yeast overnight with shaking at 30 °C in 5 mL of appropriate media ( see Note 4 ).
2. Next day, dilute the culture to A 600 ~ 0.3 to start 10 mL of secondary culture of yeast and grow to mid-log phase (A 600 ~ 0.8) ( see Note 5 ). To confi rm successful epitope tagging 5-10 transformants should be analyzed by Immuno blotting. The rate of precise integration at the desired open reading frame varies depending on the locus, but is usually around 70 %. Any protein extraction and Western blotting

Western Blotting
procedure will work to detect successful insertion of the epitope tag. Below we describe extraction using denaturing conditions, which preserves protein integrity better than many other protocols.
1. Grow transformants overnight with shaking in 1 mL of appropriate media. Also, use the parental strain without epitope tag as negative control sample. Next day, start 5-10 mL secondary culture of yeast and grow it up to A 600 ~ 0.8. Collect cells by centrifugation (20,000 × g , 1 min, room temperature), or by fi ltration. Quickly freeze the cell pellet and store it at -80 °C or process as below. 3. Separate the lysate from glass beads ( see Note 13 ). Centrifuge the lysate at 20,000 × g for 10 min at room temperature to remove cell debris and transfer the clarifi ed supernatant to a fresh microfuge tube.

Quantify protein concentration and dilute samples to 4 M
Urea using 2× SDS sample buffer. Separate 20-40 μg of protein lysate on a standard SDS polyacrylamide gel.
5. Transfer proteins onto PVDF membrane, block the membrane at room temperature with blocking buffer for 40 min with shaking and fi nally incubate the blot overnight with shaking at 4 °C with appropriate antibody, diluted at manufacture recommended dilution in blocking buffer with 0.02 % sodium azide ( see Note 14 ).
6. Wash the blot twice with 1× TBS-T ( see Note 15 ) and then incubate with secondary antibody (diluted in blocking buffer according to manufacturer's recommendations) for 1 h at room temperature with shaking.
7. Wash the blot twice with 1× TBS-T and twice with 1× TBS and then proceed to developing the blot using a chemiluminescent substrate.

Notes
1. Any other Taq polymerase kit can also be used.
2. The fi rst set of PCR cycles at 45 °C annealing temperature, ensures annealing of the short linker region of the primer to the tag cassette. The latter cycles at annealing temperature of 60 °C, allow for optimal annealing of the whole primer to the generated PCR product template.
3. Precipitation of PCR DNA is an optional step but helps to increase the effi ciency of yeast transformation.
4. Generally yeast strains should be grown in YEP media containing 2 % dextrose while strains requiring selection to retain plasmids should be grown in corresponding minimal media containing 2 % dextrose. Temperature-sensitive strains should be grown at room temperature.
5. For each transformation reaction, use 10 mL of yeast culture. Make sure to reserve one reaction for a control transformation.
6. To enhance transformation effi ciency, heat sheared salmon sperm DNA in 100 °C water bath for 10 min and then put on ice before use.

7.
A "no PCR" control transformation should also be performed with salmon sperm DNA but no PCR product. 11. Urea buffer can be made before hand and stored at room temperature. PMSF and protease inhibitors should be added fresh, prior to use.
12. Cells can be vortexed with glass beads fi ve times for 1 min at 4 °C, with a 1 min break on ice between the runs. Many other methods also exist for breaking yeast cells, such as TCA extraction, or alkali lysis.
13. A convenient way to separate the lysate from glass beads is to poke a hole at the bottom of the tube with a 21G needle, insert the tube with the hole into a fresh tube and centrifuge carefully for 30 s at 1,000 × g . The lysate will pass through the hole in the upper tube and collect in the lower tube, whereas the glass beads shall remain in the upper tube.
14. By adding sodium azide, one can increase the life of the diluted antibody. It can be used repeatedly and can be stored at −20 °C for a period of few months. Note that sodium azide should not be added to antibodies that are directly conjugated with HRP.
15. Each wash is performed for 10 min by shaking at room temperature.