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Epitopes are often used in proteomics and the study of other gene products. Using recombinant DNA techniques genetic sequences coding for epitopes that are recognized by common antibodies can be fused to the gene. Following synthesis, the resulting epitope tag allows the antibody to find the protein or other gene product enabling lab techniques for location, purification, and further molecular characterization.
Epitope tagging is accomplished by fusion of the target protein with the tag of choice. This is accomplished by insertion of the target gene into a host cell−specific expression vector that also encodes the epitope tag. Expression vectors for a variety of host cell types have been developed including Escherichia coli, yeast, insect, and mammalian cells.
Fig 1. Epitope tag protein
Epitope tags have been used in variety of applications, from Western blot analysis, immunoprecipitation, immuno-fluorescence, and immunoaffinity purification, to more recently developed approaches such as fluorescence resonance energy transfer (FRET). They have been used to determine protein size, abundance, trafficking within cells and membranes, internalization, cellular location, posttranslational modification, and interactions with other proteins. There are a number of considerations in the use of epitope tags, including the size, amino acid composition, and location of the epitope. This epitope already has a known antibody and when it is expressed in a transgenic cell it facilitates the tracking of the transgene tagged. It is superior as far as specificity to natural immunogens is concerned. Inserting tandem epitopes improves sensitivity. Generally, either the N or the C ends of the proteins are labeled. For identification, Western blots, immunoprecipitation, immunofluorescence, electron microscopy and other methods are used. Epitope tagging has wide applicability in basic proteomics and eventually to clinical problems.
The biochemical properties of commonly used epitope tags are summarized below:
Chloramphenicol acetyltransferase (CAT) tag: This 24kDa tag also serves as a reporter gene and retains its activity when fused to most proteins. This means it can be used to directly measure expression levels without the need for PAGE or immunodetection.
Dihydrofolate Reductase (DHFR) tag: This 25kDa protein is involved in the thymidine biosynthetic pathway. Purification of proteins with this tag can be achieved with methotrexate-linked resins.
FLAG tag: Charged octapeptide sequence (DYKDDDDK), useful for protein detection, especially when tandem with the 3xFLAG™ epitope (DYKDHDGDYKDHDIDYKDDDK). This helps study low-abundance proteins and optimize difficult-to-express protein projects. It's also a great tab for purification. The FLAG sequence also contains an enterokinase cleavage site, and if the tag is at the N-terminus, no excess residues are left after cleavage.
Glutathione S-transferase (GST) tag: GST was one of the first epitope tags to be used; it can be placed at the N- or C-terminus and enables soluble expression of proteins. Purification was performed using glutathione-binding resin.
Green fluorescent protein (GFP) tag: Unique among epitope tags, GFP is an autofluorescent 27 kDa tag that is directly detectable in living cells by fluorescence microscopy.
Hemagglutinin A (HA) tag: HA, derived from the binding domain of the influenza hemagglutinin protein, contains a high proportion of charged residues (YPYDVPDYA) and thus likely forms a strong antibody recognition site.
Histidine (His) tag: This is currently the most widely used purification tag; it allows purification with nickel affinity resins. These resins are resistant to denaturing conditions (useful for purification of denatured solubilized proteins from inclusion bodies) and are reusable. Binding specificity is lower than antibody resins, so additional purification steps are often required. Acid elution has been used as a low-salt substitute for imidazole, and cobalt can be used in place of nickel to improve specificity. Metalloproteins and histidine-rich proteins (such as chloramphenicol acetyltransferase) also bind to these resins, so use of appropriate controls is recommended.
Herpes Simplex Virus (HSV) tag: HSV is derived from the glycoprotein D precursor envelope protein and is short (QPELAPEDPED), so it is unlikely to interfere with protein structure or function.
Luciferase tag: Reporter gene commonly used to detect protein expression. The results can be verified by immunological methods.
Maltose binding protein (MBP) tag: The size of the tag (43kDa) contributes to its increased production of soluble proteins in E. coli. Along with GST and thioredoxin, it is also popular as a solubilizing tag. Purification is achieved using low-cost amylose resin (Asn10 spacer between tag and protein enhances resin binding).
c-Myc tag: c-Myc (or myc) has been widely used in immunoblotting, immunoprecipitation and flow cytometry. Its small size (EQKLISEEDL) means it is less likely to interfere with (or enhance) protein folding and can be placed at the N- and C-termini of proteins.
Streptavidin / Biotin epitope tag: The affinity of the streptavidin-biotin interaction (10 - 14 M) means that it can withstand harsh conditions, so it is often used to immobilize proteins (eg in the production of protein chips). Streptavidin can be fused to the protein of interest in full-length, truncated or mutated forms.
T7 epitope tag: This is a 260-residue tag of the gene 10 product from T7 bacteriophage; enhances expression levels in E. coli.
Thioredoxin epitope tag: Despite its small size (11kDa), thioredoxin is very effective as a solubilizing tag (as with all solubilizing tags, solubility does not necessarily imply functional folding, and the fusion protein may precipitate when its tag is cleaved) . Thioredoxin has the unique property of being high temperature resistant, stable at temperatures up to 80°C, and can confer certain thermostability to its fusion partners. In this way, cellular proteins can be heat denatured without affecting the fusion protein. Purification can be achieved using phenylarsine oxide resins or antibody-conjugated resins.
V5 epitope tag: Derived from amino acids 95-108 of the P/V protein of paramyxovirus SV5 (GKPIPNPLLGLDST).
Vesicular stomatitis virus glycoprotein (VSV-G) epitope tag: The amino acid sequence of VSV-G tag is (YTDIEMNRLGK).
Yeast two-hybrid tags: B42, GAL4, LexA, VP16: These tags are used for protein interactions in the yeast two-hybrid system and serve as DNA-binding (GAL4, LexA) domains; transcriptional activators (B42, GAL4, VP16)
An epitope is the part of an antigen that is recognized by an antibody. Therefore, epitope tags are often used in antibody-based assays. Epitope tags are generally shorter than affinity tags and therefore rarely affect protein function. Although they can also be used for affinity purification, immune antibody-based columns are expensive and not as efficient as affinity tags. However, epitope tags have great advantages in detection. It is widely used in cell culture and co-immunoprecipitation.
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