Common Epitope Tags(including sequence) in Protein Research

Common Epitope Tags and Sequences

 The biochemical properties of the popular epitope tags are summarized as below:

Chloramphenicol Acetyl Transferase (CAT)

This 24 kDa tag is also used as a reporter gene, and retains its functionality when fused to most proteins. This means that it can be used to measure levels of expression directly without the need for PAGE or immunodetection.

Dihydrofolate Reductase (DHFR)

This is a well-characterized 25 kDa protein involved in the thymidine biosynthesis pathway. Purification of tagged proteins can be achieved by methotrexate-linked resin.

FLAG®

The octapeptide sequence is highly charged (DYKDDDDK) and useful for sensitive detection, especially if concatenated as the 3xFLAG™ epitope (DYKDHDGDYKDHDIDYKDDDK). This facilitates the study of low-abundance proteins and the optimization of difficult protein expression projects. It is also popular as a purification tag; the high level of purification can eliminate the need for further clean-up. The FLAG sequence also contains an enterokinase cleavage site to allow removal if the tag is placed at the N-terminus.

Glutathione S-Transferase (GST)

GST was one of the first epitope tags to be used; it can be placed on the N or C-terminus and can enhance the solubility of expressed proteins. Purification is achieved with glutathione-conjugated resin.

Green Fluorescent Protein (GFP)

Unique among epitope tags, GFP is an autofluorescent 27 kDa tag that can be directly detected in living cells by fluorescent microscopy.

Hemagglutinin A (HA)

Derived from the binding domain of the Influenza hemagglutinin protein, HA contains a high proportion of charged residues (YPYDVPDYA) so it is likely to form a strong antibody recognition site.

Histidine (His)

This is by far the most widely used purification tag; it allows purification with economical nickel affinity resins. These resins tolerate denaturing conditions (useful for purifying solubilized proteins from inclusion bodies) and can be reused. Binding specificity is less than that of antibody-based resins, so a second purification step is often necessary; this also removes the high imidazole concentration used for elution. Acidic elution has been used as a low-salt alternative to imidazole, and cobalt can been used in place of nickel to increase specificity. Metalloproteins and histidine-rich proteins (e.g., Chloramphenicol Acetyltransferase) also bind to these resins, so it is advisible to use suitable controls.

Herpes Simplex Virus (HSV)

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

This is better known as a reporter gene for expression assays. Antibodies allow results to be verified by immunological methods.

Maltose-Binding Protein (MBP)

The size (43 kDa) of this tag contributes to its ability to increase soluble protein production in E. coli. Along with GST and Thioredoxin it is popular as a ‘solubility’ tag. Purification is achieved using low-cost amylose resin, an Asn10 spacer between the tag and the protein improving resin binding.

c-Myc

c-Myc (or myc) has been extensively used for Western blotting, immunoprecipitation, and flow cytometry. Its small size (EQKLISEEDL) means that it is unlikely to perturb (or enhance) protein folding and it has been used at both N and C-termini.

Protein A and Protein G

These bacterial ‘superantigens’ bind to all forms of IgG. They can be fused to proteins for purification using IgG, but they are most often employed as conjugates for generic secondary detection or purification of antibodies.

Streptavidin/Biotin

The affinity of the streptavidin-biotin interaction (10^-14 M) means that it can withstand harsh conditions, so it is popular as a method of immobilizing proteins (e.g., in production of protein arrays). Streptavidin can be incorporated as a full-length, truncated, or mutated protein, and biotin can be incorporated by fusing a protein domain that becomes biotinylated in vivo (e.g. biotin-carboxy carrier protein).

T7

This is a 260-residue tag derived from the gene 10 product of T7 phage; may enhance expression levels in E. coli.

Thioredoxin

Despite its small size (11 kDa), thioredoxin is very effective as a ‘solubilizing’ tag (As with all solubilizing tags, solubility does not necessarily mean functional folding, and fusion proteins can precipitate when their tag is cleaved). Thioredoxin is unique among epitope tags in being stable up to 80 °C and can confer some thermostability onto its fusion partner. This way, cellular proteins can be heat-denatured without affecting the fusion protein. Purification can either be achieved using phenylarsine oxide resin or antibodyconjugated resin.

V5

A small tag (GKPIPNPLLGLDST) derived from residues 95-108 of the P/V proteins of the Paramyxovirus SV5. Vectors, purification resins, and antibodies are widely available.

Vesicular Stomatitis Virus Glycoprotein (VSV-G)

The VSV-G antibody recognizes the five C-terminal residues of the tag (YTDIEMNRLGK). Like many tags, it is found as a cassette in commercially available vectors to aid cloning.

Yeast 2-hybrid tags: B42, GAL4, LexA, VP16

These tags are used to detect protein interactions in the yeast 2-hybrid system acting as DNA binding (GAL4, LexA) domains or transcriptional activators (B42, GAL4, VP16). Antibodies against these tags allow results to be validated and investigated further.

Souce: NovoPro    2018-03-28