A PCR-based strategy to generate yeast strains expressing endogenous levels of amino-terminal epitope-tagged proteins

An epitope tag introduced to a gene of interest (GOI) greatly increases the ease of studying cellular proteins. Rapid PCR-based strategies for epitope tagging a protein's C-terminus at its native gene locus are widely used in yeast. C-terminal epitope tagging is not suitable for all proteins, however. Epitope tags fused to the C-terminus can interfere with function of some proteins or can even be removed by C-terminal protein processing. To overcome such problems, proteins can be tagged with epitopes at their amino-termini, but generating yeast strains expressing N-terminal epitope tagged genes under control of the endogenous promoter at the native locus is comparatively more difficult. Strategies to introduce N-terminal epitope tags have been reported previously but often introduce additional sequences other than the epitope tag into the genome. Furthermore, N-terminal tagging of essential genes by current methods requires formation of diploid strains followed by tetrad dissection or expression of an additional copy of the GOI from a plasmid. The strategies described here provide a quick, facile means of epitope tagging the N-terminus of both essential and nonessential genes in a two-step PCR-based procedure. The procedure has the significant advantage of leaving tagged genes under the control of their endogenous promoters, and no additional sequences other than the epitope tag encoding nucleotides are inserted into the genome.     

Sequence- and position-dependent tagging protects extracellular-regulated kinase 3 protein from 26S proteasome-mediated degradation

Extracellular-regulated kinase 3, an atypical member of the mitogen-activated protein kinase subfamily of extracellular-regulated kinases, was originally identified in 1991. Little is known about the biochemical properties, regulation, and biological functions of this protein kinase, partially due to the unstable nature of endogenous and low ectopical expression level of the protein. Here, we report that a single C-terminal c-myc tag increases the half-life of ectopic expressed tagged extracellular-regulated kinase 3 approximately four times compared to the reported 30 min half-life time for the endogenous protein and ectopic expressed extracellular-regulated kinase 3 deprived of its c-myc tag. These findings indicate that this C-terminal tag stabilizes the extracellular-regulated kinase 3. The stabilizing effect of the C-terminal c-myc tag is observed in all cell types tested, but is position- and tag sequence-dependent as neither N-terminal c-myc tag nor C-terminal HA tag stabilize the protein. The c-myc tag on extracellular-regulated kinase 3 did not interfere with its kinase activity, nor did it abrogate its ability to interacts with its bona fide substrate mitogen-activated protein kinase-activated protein kinase 5, indicating that tagging did not alter the known biological properties of the protein. Stabilization of the tagged extracellular-regulated kinase 3 protein probably results from reduced ubiquitination. In conclusion, position and sequence specific tagging should provide an easy and useful tool to generate a more stable protein that can functionally substitute the endogenous unstable protein. A stabilized variant may facilitate studies on the biological role of the protein.     

Functional expression of N-terminally tagged membrane bound cytochrome P450

The introduction of an affinity tag offers an attractive approach to isolation of membrane proteins. The type of affinity tag and its positioning in the protein is determined by the desired subsequent experimental uses of the isolated protein. To minimize the risk of interference, membrane proteins may preferentially be tagged on the side of the membrane that does not harbor the active site. In cytochromes P450, affinity tags have traditionally been introduced at the C-terminal to obtain high expression levels and to avoid translocation of the affinity tag over the membrane bilayer. Using the plant cytochrome P450 CYP79A1 and CYP71E1 as model systems, we demonstrate that a full-length CYP79A1 strepII tagged at the N-terminal expresses well and is able to translocate over the lipid bilayer to produce a functionally active protein that is amenable to affinity purification. The expression level and activity of the N-terminally tagged CYP79A1 protein are very similar to those obtained for the C-terminally tagged version. As an experimental tool, ER luminal tagging is envisioned to offer many advantages in future P450 research work e.g. when catalytic properties of an enzyme or protein–protein interactions are to be investigated.     

Biotin tagging coupled with amino acid‐coded mass tagging for efficient and precise screening of interaction proteome in mammalian cells

In mammalian cells, when tandem affinity purification approach is employed, the existence of untagged endogenous target protein and repetitive washing steps together result in overall low yield of purified/stable complexes and the loss of weakly and transiently interacting partners of biological significance. To avoid the trade‐offs involving in methodological sensitivity, precision, and throughput, here we introduce an integrated method, biotin tagging coupled with amino acid‐coded mass tagging, for highly sensitive and accurate screening of mammalian protein–protein interactions. Without the need of establishing a stable cell line, using a short peptide tag which could be specifically biotinylated in vivo, the biotin‐tagged target/bait protein was then isolated along with its associates efficiently by streptavidin magnetic microbeads in a single step. In a pulled‐down complex amino acid‐coded mass tagging serves as “in‐spectra” quantitative markers to distinguish those bait‐specific interactors from non‐specific background proteins under stringent criteria. Applying this biotin tagging coupled with amino acid‐coded mass tagging approach, we first biotin‐tagged in vivo a multi‐functional protein family member, 14‐3‐3ε, which was expressed at close to endogenous level. Starting with approximately 20 millions of 293T cells which were significantly less than what needed for a tandem affinity purification run, 266 specific interactors of 14‐3‐3ε were identified in high confidence.     

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.     

Telemetry tag effects on juvenile lingcod Ophiodon elongatus movement: a laboratory and field study

This study tested the behavioural effects of tagging subyearling and yearling lingcod Ophiodon elongatus with acoustic telemetry tags in laboratory tanks and in the natural environment (Puget Sound, WA). In the laboratory, tagged individuals showed less movement and feeding behaviour soon after tagging than untagged controls. The effect dissipated after c. 1 week, presumably as the tagged O. elongatus recovered from surgery or adjusted to the presence of the tags. This dissipation enabled a field study that compared early‐tagged individuals with a long recovery period after tagging to recently‐tagged individuals with a short recovery period after tagging. Consistent with findings from the laboratory experiment, recently tagged individuals showed less movement away from three release sites in Puget Sound than early‐tagged individuals. Together, the laboratory and field results provide evidence of temporary tag effects on actual movement in the natural environment and provide a method for testing tag effects in the field. This study suggests that subyearling and yearling O. elongatus should be held for a recovery period before release. If holding after tagging is not an option, then movement data collected during the first week should be interpreted cautiously.     

Assessment of flipper tag site healing in gray seal pups using thermography

Infrared thermography was used to monitor the healing process at flipper tag sites in gray seal (Halichoerus grypus) pups. We tested the hypothesis that tagging would result in a rise in surface temperature associated with tag site healing processes compared with adjacent untagged areas of the flipper. Prior to tagging thermal images were recorded of the dorsal side of hind flippers of pups tagged in early lactation (n= 20) and at weaning (n= 19) on the Isle of May, Scotland (56°11′N, 02°33′W) from October to December 2008. Pups tagged in early lactation were sampled again at late lactation, at weaning and then every 3 d for an average of 29 d post‐tagging while pups tagged at weaning were sampled every 3 d for an average of 17 d post‐tagging. Tag sites were also scored for signs of infection or swelling at each sampling. Results showed that (1) small temperature increases associated with wound healing processes around the tag site returned to pre‐tagging levels before animals leave the island and (2) there was little evidence of tagging‐related infections or tag loss irrespective of age at tagging.     

Modules for C-terminal epitope tagging of Tetrahymena genes

Although epitope tagging has been widely used for analyzing protein function in many organisms, there are few genetic tools for epitope tagging in Tetrahymena. In this study, we describe several C-terminal epitope tagging modules that can be used to express tagged proteins in Tetrahymena cells by both plasmid- and PCR-based strategies.     

Epitope-tagged ubiquitin. A new probe for analyzing ubiquitin function.

In the present work, a method based on an epitope-tagged ubiquitin derivative is described that allows for the unambiguous detection of ubiquitin-protein conjugates formed in vivo or in vitro. Expression in the yeast Saccharomyces cerevisiae of ubiquitin that has been tagged at its amino terminus with a peptide epitope results in the formation of tagged ubiquitin-protein conjugates that are detectable by immunoblotting with a monoclonal antibody that recognizes the tag. The expression of tagged ubiquitin has no adverse effect on vegetative growth and, moreover, can suppress the stress-hypersensitive phenotype of yeast lacking the polyubiquitin gene UBI4. We also show that tagged ubiquitin is correctly conjugated in vivo and in vitro to a short-lived test protein and can be covalently extended into the multimeric ubiquitin chain that is normally required for the degradation of this protein. Surprisingly, however, conjugation of tagged ubiquitin inhibits proteolysis. These and related results suggest that the amino-terminal region of ubiquitin is important in protease-substrate recognition and that the multiubiquitin chain is a dynamic transient structure. The potential of tagged ubiquitin for the identification and isolation of ubiquitin-protein conjugates and ubiquitin-related enzymes, and as a tool in mechanistic studies is discussed.     

Influence of V5/6-His Tag on the Properties of Gap Junction Channels Composed of Connexin43, Connexin40 or Connexin45

HeLa cells expressing wild-type connexin43, connexin40 or connexin45 and connexins fused with a V5/6-His tag to the carboxyl terminus (CT) domain (Cx43-tag, Cx40-tag, Cx45-tag) were used to study connexin expression and the electrical properties of gap junction channels. Immunoblots and immunolabeling indicated that tagged connexins are synthesized and targeted to gap junctions in a similar manner to their wild-type counterparts. Voltage-clamp experiments on cell pairs revealed that tagged connexins form functional channels. Comparison of multichannel and single-channel conductances indicates that tagging reduces the number of operational channels, implying interference with hemichannel trafficking, docking and/or channel opening. Tagging provoked connexin-specific effects on multichannel and single-channel properties. The Cx43-tag was most affected and the Cx45-tag, least. The modifications included (1) V _j-sensitive gating of I _j (V _j, gap junction voltage; I _j, gap junction current), (2) contribution and (3) kinetics of I _j deactivation and (4) single-channel conductance. The first three reflect alterations of fast V _j gating. Hence, they may be caused by structural and/or electrical changes on the CT that interact with domains of the amino terminus and cytoplasmic loop. The fourth reflects alterations of the ion-conducting pathway. Conceivably, mutations at sites remote from the channel pore, e.g., 6-His-tagged CT, affect protein conformation and thus modify channel properties indirectly. Hence, V5/6-His tagging of connexins is a useful tool for expression studies in vivo. However, it should not be ignored that it introduces connexin-dependent changes in both expression level and electrophysiological properties.     

Functional properties and modulation of extracellular epitope - tagged CaV2.1 voltage-gated calcium channels

Depolarisation-induced Ca²⁺ influx into electrically excitable cells is determined by the density of voltage-gated Ca²⁺ channels at the cell surface. Surface expression is modulated by physiological stimuli as well as by drugs and can be altered under pathological conditions. Extracellular epitope tagging of channel subunits allows to quantify their surface expression and to distinguish surface channels from those in intracellular compartments. Here we report the first systematic characterisation of extracellularly epitope tagged Ca_V2.1 channels. We identified a permissive region in the pore-loop of repeat IV within the Ca_V2.1 α1 subunit which allowed integration of several different tags (hemagluttinine [HA], double HA; 6-histidine tag [His], 9-His, bungarotoxin-binding site) without compromising α1 subunit protein expression (in transfected tsA-201 cells) and function (after expression in X. laevis oocytes). Immunofluorescent studies revealed that the double-HA tagged construct (1722-HAGHA) was targeted to presynaptic sites in transfected cultured hippocampal neurons as expected for Ca_V2.1 channels. We also demonstrate that introduction of tags into this permissive position creates artifical sites for channel modulation. This was demonstrated by partial inhibition of 1722-HA channel currents with anti-HA antibodies and the concentration-dependent stimulation or partial inhibition by Ni-nitrilo triacetic acid (NTA) and novel bulkier derivatives (Ni-trisNTA, Ni-tetrakisNTA, Ni-nitro-o-phenyl-bisNTA, Ni-nitro-p-phenyl-bisNTA). Therefore our data also provide evidence for the concept that artificial modulatory sites for small ligands can be introduced into voltage-gated Ca²⁺ channel for their selective modulation.     

pAUL: A Gateway-Based Vector System for Adaptive Expression and Flexible Tagging of Proteins in Arabidopsis

Determination of protein function requires tools that allow its detection and/or purification. As generation of specific antibodies often is laborious and insufficient, protein tagging using epitopes that are recognized by commercially available antibodies and matrices appears more promising. Also, proper spatial and temporal expression of tagged proteins is required to prevent falsification of results. We developed a new series of binary Gateway cloning vectors named pAUL1-20 for C- and N-terminal in-frame fusion of proteins to four different tags: a single (i) HA epitope and (ii) Strep-tagIII, (iii) both epitopes combined to a double tag, and (iv) a triple tag consisting of the double tag extended by a Protein A tag possessing a 3C protease cleavage site. Expression can be driven by either the 35 S CaMV promoter or, for C-terminal fusions, promoters from genes encoding the chloroplast biogenesis factors HCF107, HCF136, or HCF173. Fusions of the four promoters to the GUS gene showed that endogenous promoter sequences are functional and drive expression more moderately and consistently throughout different transgenic lines when compared to the 35 S CaMV promoter. By testing complementation of mutations affected in chloroplast biogenesis factors HCF107 and HCF208, we found that the effect of different promoters and tags on protein function strongly depends on the protein itself. Single-step and tandem affinity purification of HCF208 via different tags confirmed the integrity of the cloned tags.     

Minimal FLAG sequence useful in the functional epitope tagging of H-Ras

Epitope tagging can interfere with normal protein function, indicating the need for an unobtrusive epitope tag. The FLAG epitope (DYKDDDDK) was examined for a minimal epitope useful in the tagging of H-Ras. The heptapeptide tag, F7 (MDYKDDD), was found to retain reactivity with M2 and M5 monoclonal antibodies in immunoprecipitation, Western blotting, and immunofluorescence microscopy. The F7 tag did not interfere with Ras stability, EGF stimulation of Ras activation, and downstream phosphorylation of MAPK Erk1/2. Unlike the full FLAG sequence, the F7 tag had minimal effect on the growth properties of H-Ras in a colony-forming assay. The F7 tag may be useful when minimizing the effect of tagging on protein function is an important criterion in the selection of an N-terminal epitope tag.     

An evaluation of elastomer and coded wire tag performance in juvenile Tibet fish Oxygymnocypris stewartii (Lloyd, 1908) under laboratory conditions

A 95‐day experiment was conducted to evaluate the performance of a visible implant elastomer tag (VIE) versus a coded wire tag (CWT) implanted in juvenile Tibet fish Oxygymnocypris stewartii (Lloyd, 1908; total length 5~7 cm) under laboratory conditions. Mortality, tag retention and growth in three groups of juvenile O. stewartii (VIE‐tagged, CWT‐tagged and control) in duplicate were determined in six indoor tanks (300‐L/tank volume, 100 fish/tank) at 15.6 ± 0.5°C water temperature. Results showed that neither tagging method had a significant difference on the mortality of the experimental fish, but that the growth rate in the VIE group was significantly lower than in the CWT and control groups. Mean tag retention in the VIE group was 95.2%, and 98.9% in the CWT group, with no significant differences in tag retention in the two methods. The study indicates that both VIE and CWT are suitable short‐term tagging methods for hatchery O. stewartii juveniles.     

A useful epitope tag derived from maltose binding protein

Maltose binding protein (MBP) is used in recombinant protein expression as an affinity and solubility tag. The monoclonal antibody B48 binds MBP tightly and has no cross‐reactivity to other proteins in an Escherichia coli lysate. This high level of specificity suggested that MBP contains an epitope that could prove useful as a purification and visualization tag for proteins expressed in E. coli. To discover the MBP epitope, a co‐crystal structure was determined for MBP bound to its antibody and four amino acids of MBP were identified as critical for the binding interaction. Fusions of various fragments of MBP to the glutathione S‐transferase protein were engineered in order to identify the smallest fragment still recognized by the α‐MBP antibody. Stabilization of the epitope via mutational engineering resulted in a minimized 14 amino‐acid tag.     

Purification and Functional Reconstitution of the Human CHIP28 Water Channel Expressed inSaccharomyces cerevisiae

The yeastSaccharomyces cerevisiaewas used for heterologous expression of the human CHIP28 water Aquaporin-1 channel (Aquaporin-1). A nine-amino-acid epitope of the influenza hemagglutinin protein (HA epitope), recognized by the monoclonal antibody 12CA5, was chosen to tag CHIP28 at its N-terminus. Epitope-tagged CHIP28 was purified from yeast extracts by immunochromatography on protein A/12CA5-coupled beads, after KI extraction and detergent solubilization, then concentrated by anion exchange chromatography. Purified protein was reconstituted in proteoliposomes and was shown to function as a water channel by stopped-flow spectrophotometry. This study demonstrates that the yeast has the capacity to produce functional aquaporins at levels sufficient for biochemical and biophysical analyses.