New modules for the repeated internal and N-terminal epitope tagging of genes in Saccharomyces cerevisiae

Epitope tagging is a powerful method for the rapid analysis of protein function. In Saccharomyces cerevisiae epitope tags are introduced easily into chromosomal loci by homologous recombination using a simple PCR-based strategy. Although quite a number of tools exist for C-terminal tagging as well as N-terminal tagging of proteins expressed by heterologous promoters, there are only very limited possibilities to tag proteins at the N-terminus and retain the endogenous expression level. Furthermore, no PCR-templates for internal tagging have been reported. Here we describe new modules that are suitable for both the repeated N-terminal and internal tagging of proteins, leaving their endogenous promoters intact. The tags include 6xHA, 9xMyc, yEGFP, TEV-GST-6xHIS, ProtA, TEV-ProtA and TEV-ProtA-7xHIS in conjunction with different heterologous selection markers.     

A vector system for efficient and economical switching of C-terminal epitope tags in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, one-step PCR-mediated modification of chromosomal genes allows fast and efficient tagging of yeast proteins with various epitopes at the C- or N-terminus. For many purposes, C-terminal tagging is advantageous in that the expression pattern of epitope tag is comparable to that of the authentic protein and the possibility for the tag to affect normal folding of polypeptide chain during translation is minimized. As experiments are getting complicated, it is often necessary to construct several fusion proteins tagged with various kinds of epitopes. Here, we describe development of a series of plasmids that allow efficient and economical switching of C-terminally tagged epitopes, using just one set of universal oligonucleotide primers. Containing a variety of epitopes (GFP, TAP, GST, Myc, HA and FLAG tag) and Kluyveromyces lactis URA3 gene as a selectable marker, the plasmids can be used to replace any MX6 module-based C-terminal epitope tag with one of the six epitopes. Furthermore, the plasmids also allow additional C-terminal epitope tagging of proteins in yeast cells that already carry MX6 module-based gene deletion or C-terminal epitope tag.     

Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe

We describe the construction of tagging cassettes and plasmids for tandem affinity purification (TAP) of proteins in Schizosaccharomyces pombe. The tagging cassettes are designed for either carboxy- or amino-terminal tagging of proteins. The carboxyl terminal tags differ in that they contain either two or four repeats of IgG binding units. For tagging endogenous loci, the cassettes contain the kan MX6 module to allow for selection of G418-resistant cells. The amino-terminal tagging vectors allow for the regulated expression of proteins. Sz. pombe Cdc2p was chosen to test these new affinity tags. Several known binding proteins co-purified with both Cdc2p-CTAP and N-TAP-Cdc2p, indicating the usefulness of these tags for the rapid purification of stable protein complexes from Sz. pombe.     

Additional vectors for PCR-based gene tagging in Saccharomyces cerevisiae and Schizosaccharomyces pombe using nourseothricin resistance

The one-step PCR-mediated technique used for modification of chromosomal loci is a powerful tool for functional analysis in yeast. Both Saccharomyces cerevisiae and Schizosaccharomyces pombe are amenable to this technique. However, the scarce availability of selectable markers for Sz. pombe hampers the easy use of this technique in this species. Here, we describe the construction of new vectors deriving from the pFA6a family, which are suitable for tagging in both yeasts owing to the presence of a nourseothricin-resistance cassette. These plasmids allow various gene manipulations at chromosomal loci, viz. N- and C-terminal tagging with 3HA (haemagglutinin) or 13Myc epitopes, GST (glutathione S-transferase), 4TAP (tandem affinity purification) and several GFP (green fluorescent protein) isoforms. For N-terminal modifications, the use of different promoters allows constitutive (PADH1) or regulatable (PGAL1) promoters for S. cerevisiae and derivatives of Pnmt1 for Sz. pombe expression.     

pDUAL, a multipurpose, multicopy vector capable of chromosomal integration in fission yeast

A novel series of plasmid vectors named pDUAL have been developed. These vectors enable one to introduce not only multicopies of genes with episomal maintenance but also a single copy with chromosomal integration into the fission yeast, Schizosaccharomyces pombe. The multicopy plasmids can be easily converted to fragments for chromosomal integration by digestion of the plasmids with a certain restriction endonuclease before transformation of the yeast cells. The resultant fragments, lacking the autonomously replicating sequence, are designed for targeting into the chromosomal leu1 locus by homologous recombination. Whether the transformants are the results of episomal maintenance of the plasmid or homologous gene targeting can be readily checked by their requirement for uracil or leucine, or by the PCR diagnostic analysis. Furthermore, we propose the use of pDUAL derivatives for PCR-based chromosomal tagging of a gene to introduce several tags into 5'-terminus of a gene, employing a set of primers. Using these all-in-one vectors, a suitable mode of expression of a cloned gene can be selected for individual analysis without any complicated subcloning processes.     

Expanding the repertoire of plasmids for PCR-mediated epitope tagging in yeast

Epitope tagging of yeast proteins provides a convenient means of tracking proteins of interest in Western blots and immunoprecipitation experiments without the need to raise and test specific antibodies. We have constructed four plasmids for use as templates in PCR-based epitope tagging in the yeast Saccharomyces cerevisiae. These plasmids expand the range of epitopes available in a tag-URA3-tag context to include the FLAG, HSV, V5 and VSV-G epitopes. The cloning strategy used would be easily applicable to the construction of a similar tag-URA3-tag molecule for essentially any desired epitope. Oligonucleotides designed for PCR from one plasmid may be used interchangeably with any of the other template molecules to allow tagging with different epitopes without the need for new primer synthesis. We have tagged Tfc6 with each of the triple epitope tags and assessed the efficiency of these epitopes for chromatin immunoprecipitation (ChIP). For all the tagged alleles, ChIP occupancy signals are easily detectable at known Tfc6 target genes. These new tags provide additional options in experimental schemes requiring multiple tagged proteins.     

Cassettes for PCR-mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans.

We have developed a set of plasmids containing fluorescent protein cassettes for use in PCR-mediated gene tagging in Candida albicans. We engineered YFP and CFP variants of the GFP sequence optimized for C. albicans codon usage. The fluorescent protein sequences, linked to C. albicans auxotrophic marker sequences, were amplified by PCR and transformed directly into yeast. Gene-specific sequence was incorporated into the PCR primers, such that the tag-cassette integrates by homologous recombination at the 3'-end of the gene of interest. This technique was used to tag Cdc3 and Tub1 with GFP, YFP and CFP, which were readily visualized by fluorescence microscopy and localized as expected. In addition, Tub1-YFP and Cdc3-CFP were visualized in the same cells. Thus, this technique directs one-step construction of multiple fluorescent protein fusions, facilitating the study of protein co-expression and co-localization in C. albicans cells in vivo.     

New drug-resistant cassettes for gene disruption and epitope tagging in Schizosaccharomyces pombe

We describe new heterologous modules for PCR-based gene targeting in the fission yeast Schizosaccharomyces pombe. Two bacterial genes, hph and nat, which display dominant drug-resistance phenotypes, are used as new selectable markers in these modules. Both genes have been used successfully in the budding yeast Saccharomyces cerevisiae, in which hph confers resistance to hygromycin B, while nat confers nourseothricin resistance (Goldstein and McCusker, 1999). Vector modules for gene disruption and C-terminal tagging with 3HA, 13Myc and GFP(S65T) are constructed using previously constructed pFA6a-MX6-derived plasmids (B?hler et al., 1998; Wach et al., 1997). In combination with the existing systems that are based upon the G418-resistance gene (kan), triple gene deletions or tags could be constructed. In addition a vector for one-step integration of a monomeric RFP (mRFP) to the C-terminus of proteins of interest is developed. Finally, oligonucleotides that allow a simple marker switch from kan to hph or nat, and vice versa, are described. The new constructs developed here should facilitate post-genomic molecular analysis of protein functions in fission yeast.     

N‐ICE plasmids for generating N‐terminal 3 × FLAG tagged genes that allow inducible,constitutive or endogenous expression in Saccharomyces cerevisiae

PCR‐mediated homologous recombination is a powerful approach to introduce epitope tags into the chromosomal loci at the N‐terminus or the C‐terminus of targeted genes. Although strategies of C‐terminal epitope tagging of target genes at their loci are simple and widely used in yeast, C‐terminal epitope tagging is not practical for all proteins. For example, a C‐terminal tag may affect protein function or a protein may get cleaved or processed, resulting in the loss of the epitope tag. Therefore, N‐terminal epitope tagging may be necessary to resolve these problems. In some cases, an epitope tagging strategy is used to introduce a heterologous promoter with the epitope tag at the N‐terminus of a gene of interest. The potential issue with this strategy is that the tagged gene is not expressed at the endogenous level. Another strategy after integration is to excise the selection marker, using the Cre‐LoxP system, leaving the epitope tagged gene expressed from the endogenous promoter. However, N‐terminal epitope tagging of essential genes using this strategy requires a diploid strain followed by tetrad dissection. Here we present 14 new plasmids for N‐terminal tagging, which combines two previous strategies for epitope tagging in a haploid strain. These ‘N‐ICE’ plasmids were constructed so that non‐essential and essential genes can be N‐terminally 3 × FLAG tagged and expressed from an inducible promoter (GAL1), constitutive promoters (CYC1 or PYK1) or the endogenous promoter. We have validated the N‐ICE plasmid system by N‐terminal tagging two non‐essential genes (SET1 and SET2) and two essential genes (ERG11 and PKC1). Copyright © 2016 John Wiley & Sons, Ltd.