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.     

HB tag modules for PCR-based gene tagging and tandem affinity purification in Saccharomyces cerevisiae

We have recently developed the HB tag as a useful tool for tandem-affinity purification under native as well as fully denaturing conditions. The HB tag and its derivatives consist of a hexahistidine tag and a bacterially-derived in vivo biotinylation signal peptide, which support sequential purification by Ni2+ -chelate chromatography and binding to immobilized streptavidin. To facilitate tagging of budding yeast proteins with HB tags, we have created a series of plasmids with various selectable markers. These plasmids allow single-step PCR-based tagging and expression under control of the endogenous promoters or the inducible GAL1 promoter. HB tagging of several budding yeast ORFs demonstrated efficient biotinylation of the HB tag in vivo by endogenous yeast biotin ligases. No adverse effects of the HB tag on protein function were observed. The HB tagging plasmids presented here are related to previously reported epitope-tagging plasmids, allowing PCR-based tagging with the same locus-specific primer sets that are used for other widely used epitope-tagging strategies. The Sequences for the described plasmids were submitted to GenBank under Accession Numbers DQ407918-pFA6a-HBH-kanMX6 DQ407927-pFA6a-RGS18H-kanMX6 DQ407919-pFA6a-HBH-hphMX4 DQ407928-pFA6a-RGS18H-hphMX4 DQ407920-pFA6a-HBH-TRP1 DQ407929-pFA6a-RGS18H-TRP1 DQ407921-pFA6a-HTB-kanMX6 DQ407930-pFA6a-kanMX6-PGAL1-HBH DQ407922-pFA6a-HTB-hphMX4 DQ407931-pFA6a-TRP1-PGAL1-HBH DQ407923-pFA6a-HTB-TRP1 DQ407924-pFA6a-BIO-kanMX6 DQ407925-pFA6a-BIO-hphMX4 DQ407926-pFA6a-BIO-TRP1.     

A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes

Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR-primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C-terminal tagged genes or for the exchange of the promoter and N-terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C-terminal epitope tags as well as nine different promoter substitutions in combination with N-terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast-optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N- and C-terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost-effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches.     

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.     

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.     

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.     

Additional cassettes for epitope and fluorescent fusion proteins in Candida albicans

Epitope tags that confer specific properties, including affinity for resins or antibodies or detection by fluorescence microscopy, are highly useful for biochemical and cell biological investigations. In Candida albicans and several other related yeasts, the CUG codon specifies serine instead of leucine, requiring that molecular tools be customized for use in this important human fungal pathogen. Here we report the construction of a set of plasmids containing 13‐Myc, 3HA, GST, V5 or His9 epitope cassettes that facilitate PCR‐mediated construction of epitope‐tagged proteins. Common primer sets amplify the different tags with two different selectable markers. In addition, we report construction of a codon‐optimized Discosoma red fluorescent protein (DsRFP) gene. Like mCherryRFP, this DsRFP signal is detectable in transformants at the colony level and is useful in double‐labelling experiments with green fluorescent protein (GFP). Finally, we describe a construct that directs PCR‐mediated two‐step insertion of GFP internal to a coding sequence, which facilitates tagging of secreted proteins, including GPI‐anchor cell wall proteins that require endogenous N‐ and C‐termini for function. These reagents expand the repertoire of molecular tools available for working with C. albicans and other members of the CUG clade of pathogenic yeasts. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Targeting of heterologous membrane proteins into proliferated internal membranes in Saccharomyces cerevisiae

Overproduction of chimeric proteins containing the HMG2/1 peptide, which comprises the seven transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl-CoA reductase isozymes 1 and 2, has previously been observed to induce the proliferation of internal endoplasmic reticulum-like membranes. In order to exploit this amplified membrane surface area for the accommodation of heterologous microsomal proteins, we fused sequences coding for human cytochrome P4501A1 (CYP1A1) to sequence encoding the HMG2/1 peptide and expressed the hybrid genes in yeast. The heterologous hybrid proteins were targeted into strongly proliferated membranes, as shown by electron microscopic and immunofluorescent analysis. Fusion proteins comprising the whole CYP1A1 polypeptide (HMG2/1-CYP1A1) exhibited 7-ethoxyresorufin-O-deethylase activity, whereas fusion proteins lacking the N-terminal 56 amino acids of CYP1A1 (HMG2/1-ΔCYP1A1) were inactive and appeared to be unable to incorporate protoheme. Similar amounts of heterologous protein were detected in cells expressing HMG2/1-CYP1A1, HMG2/1-ΔCYP1A1 and CYP1A1, respectively. Replacement of the N-terminal membrane anchor domain of human NADPH-cytochrome P450 oxidoreductase by the HMG2/1 peptide also resulted in a functional fusion enzyme, which was able to interact with HMG2/1-CYP1A1 and the yeast endogenous P450 enzyme lanosterol-14α-demethylase.     

Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae

Green fluorescent protein (GFP) has become an increasingly popular protein tag for determining protein localization and abundance. With the availability of GFP variants with altered fluorescence spectra, as well as GFP homologues from other organisms, multi-colour fluorescence with protein tags is now possible, as is measuring protein interactions using fluorescence resonance energy transfer (FRET). We have created a set of yeast tagging vectors containing codon-optimized variants of GFP, CFP (cyan), YFP (yellow), and Sapphire (a UV-excitable GFP). These codon-optimized tags are twice as detectable as unoptimized tags. We have also created a tagging vector containing the monomeric DsRed construct tdimer2, which is up to 15-fold more detectable than tags currently in use. These tags significantly improve the detection limits for live-cell fluorescence imaging in yeast, and provide sufficient distinguishable fluorophores for four-colour imaging.     

Plasmids with E2 epitope tags: tagging modules for N‐ and C‐terminal PCR‐based gene targeting in both budding and fission yeast,and inducible expression vectors for fission yeast

A single‐step PCR‐based epitope tagging enables fast and efficient gene targeting with various epitope tags. This report presents a series of plasmids for the E2 epitope tagging of proteins in Saccharomyces cerevisiae and Schizosaccharomyces pombe. E2Tags are 10‐amino acids (epitope E2a: SSTSSDFRDR)‐ and 12 amino acids (epitope E2b: GVSSTSSDFRDR)‐long peptides derived from the E2 protein of bovine papillomavirus type 1. The modules for C‐terminal tagging with E2a and E2b epitopes were constructed by the modification of the pYM‐series plasmid. The N‐terminal E2a and E2b tagging modules were based on pOM‐series plasmid. The pOM‐series plasmids were selected for this study because of their use of the Cre–loxP recombination system. The latter enables a marker cassette to be removed after integration into the loci of interest and, thereafter, the tagged protein is expressed under its endogenous promoter. Specifically for fission yeast, high copy pREP plasmids containing the E2a epitope tag as an N‐terminal or C‐terminal tag were constructed. The properties of E2a and E2b epitopes and the sensitivity of two anti‐E2 monoclonal antibodies (5E11 and 3F12) were tested using several S. cerevisiae and Sz. pombe E2‐tagged strains. Copyright © 2009 John Wiley & Sons, Ltd.     

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

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 × 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 ). Copyright © 2009 John Wiley & Sons, Ltd.     

Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae

Epitope tagging is the insertion of a short stretch of amino acids constituting an epitope into another protein. Tagged proteins can be identified by Western, immunoprecipitation and immunofluorescence assays using pre-existing antibodies. We have designed vectors containing the URA3 gene flanked by direct repeats of epitope tags. We use the polymerase chain reaction (PCR) to amplify the tag-URA3-tag cassette such that the ends of the PCR fragments possess homology to the gene of interest. In vivo recombination is then used to direct integration of the fragment to the location of interest, and transformants are selected by their Ura⁺ phenotype. Finally, selection for Ura^? cells on 5-fluoro-orotic acid plates yields cells where recombination between the repeated epitopes has ‘popped out’ the URA3 gene, leaving a single copy of the epitope at the desired location. PCR epitope tagging (PET) provides a rapid and direct technique for tagging that does not require any cloning steps. We have used PET to tag three Saccharomyces cerevisiae proteins, Cln1, Sic1 and Est1.     

Photoactivatable GFP tagging cassettes for protein-tracking studies in the budding yeast Saccharomyces cerevisiae

Yeast cell biologists use a variety of fluorescent protein tags for determining protein localization and for measuring protein dynamics using fluorescence recovery after photobleaching (FRAP). Although many modern fluorescent proteins, such as those with photoactivatable and photoconvertible characteristics, have been developed, none has been exploited for studies in budding yeast. We describe here the construction of yeast-tagging vectors containing photoactivatable green fluorescent protein (PA-GFP) for analysis of protein behaviour. We tagged two yeast proteins, Erg6p and Num1p, with PA-GFP and demonstrated specific photoactivation of the fusion proteins in live cells. Fluorescence intensity measurements showed that a short 5 s exposure to 413 nm light is sufficient to produce the maximum level of activated GFP fluorescence. Local photoactivation of cortical Num1p-PA-GFP showed movement of the marked proteins, providing new insights into the behaviour of Num1p at the cell cortex. Since photoactivation can be achieved using standard mercury arc illumination, the PA-GFP tag represents a convenient and economical way to determine protein dynamics in the cell. Thus, the tagging modules should facilitate protein-tracking studies in a wide variety of cell biological processes in yeast.     

乳酸乳球菌表达系统及其启动子的研究进展

乳酸乳球菌（Lactococcus lactis）是工业应用中一种重要的模式菌株，具有非致病性、分泌蛋白能力强、容易分离培养等特性，是异源蛋白表达和分泌的理想宿主。高效可控的启动子是实现外源蛋白高效表达的关键因素之一。根据诱导机制，启动子可分为组成型和诱导型。本文阐述了乳酸乳球菌表达系统及其启动子结构，总结了生物信息学预测启动子及筛选克隆新启动子的方法，并对乳酸乳球菌启动子未来的研究方向进行了展望，可为深入研究启动子的结构和功能并进一步在工业上生产外源蛋白提供参考。     

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.     

An expanded tool kit for the auxin‐inducible degron system in budding yeast

Fusion of inducible degradation signals, so‐called degrons, to cellular proteins is an elegant method of controlling protein levels in vivo. Recently, a degron system relying on the plant hormone auxin has been described for use in yeast and vertebrate cells. We now report the construction of a series of vectors that significantly enhance the versatility of this auxin‐inducible degron (AID) system in Saccharomyces cerevisiae. We have minimized the size of the degron and appended a series of additional epitope tags, allowing detection by commercial antibodies or fluorescence microscopy. The vectors are compatible with PCR‐based genomic tagging strategies, allow for C‐ or N‐terminal fusion of the degron, and provide a range of selection markers. Application to a series of yeast proteins, including essential replication factors, provides evidence for a general usefulness of the system. © 2013 The Authors. Yeast published by John Wiley & Sons, Ltd.