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Epitope tagging
Bill Brizzard
Indiana University Research and Technology Corporation, Bloomington, Indiana
BioTechniques 25th Anniversary, Vol. 44, No. 5, April 2008, pp. 693–695
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Epitope tagging is widely used in the characterization of newly discovered proteins. This review presents an overview of how the technique evolved and how it is being used today, with a focus on its use in the study of protein-protein interactions. In addition, the evolution of the technique for proteomic analyses is described.


Epitope tagging is a technique in which a known epitope is fused to a recombinant protein by means of genetic engineering. By choosing an epitope for which an antibody is available, the technique makes it possible to detect proteins for which no antibody is available. This is especially useful for the characterization of newly discovered proteins and proteins of low immunogenicity. By selection of the appropriate epitope and antibody pair, it is possible to find a combination with properties that are suitable for the desired experimental application, such as Western blot analysis, immunoprecipitation, immunochemistry, and affinity purification.

How It Started

The advent of hybridoma technology made monoclonal antibodies available, thereby providing a reproducible source of antibody. However, the first commercially available epitope tags were originally designed for protein purification. Examples of these early commercial products include the FLAG, 6 × His, and glutathione-S-transferase (GST) systems. The FLAG tagging system in its original version included the anti-FLAG M1 monoclonal antibody with calcium-dependent binding. FLAG-tagged proteins can be eluted from the M1 antibody with EDTA (1). Likewise, the 6 × His tag is used for purification of recombinant proteins by means of metal chelate chromatography (2). Similarly, GST-tagged proteins can be purified using glutathione agarose (3). In addition to the commercial tags, the development of the other tags such as HA (4) and c-myc (5) were reported by academic groups. As research progressed and recombinant DNA technology evolved, the utility of epitope tags in the study of protein interaction was recognized. The result was rapid growth in the number of available tag and antibody pairs. For example, the anti-FLAG M2 monoclonal antibody was made available commercially (6) as were monoclonal antibodies for the 6 × His tag (7), HA tag, and c-myc tag.

Where It Stands

There are now numerous types of tags with different features suited to diverse applications. The tags discussed in this review are listed in (Table 1). For information on additional tags, see Reference 8,. The most significant of these was the discovery of fluorescent protein reporters (9), which made it possible to detect proteins intracellularly without the need of a secondary reagent. The numerous applications of fluorescent reporters are beyond the scope of this review. While the use of epitope-tagging facilitates the study and characterization of newly discovered proteins, the technique does have some limitations. Notably, the insertion of an epitope tag can alter protein function. For a discussion of the effects of tag location on protein function, see Reference 8,.

Table 1. Common Epitope Tags

GST, glutathione-S-transferase; CBP, calmodulin-binding peptide.

In addition to the traditional antibody and epitope combinations, other types of affinity tags have been discovered. An early example is the protein A tag, which binds to IgG (10). Other examples include those based on interaction with streptavidin (11) and biotin (12), maltose-binding peptide (MBP tag) and maltose (13), chitin-binding domain (CBD) and chitin (14), and the calmodulin-binding peptide that binds to calmodulin (15). Another type of tag is the S-peptide tag that binds to the S-protein derived from pancreatic RNase A (16).

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. In many cases, the recognition sequence for a protease is included after the coding sequence of the epitope tag to allow for release of the epitope tag. In the case of the FLAG tag, the epitope includes the recognition sequence for the protease enterokinase. Another option is use of self-cleaving tags in which a self-cleaving intein fused to the CBD tag is used (14). Vectors are available that allow for N-terminal and C-terminal tagging. The discovery of PCR greatly facilitated creation of tagged proteins by making it possible to fuse the desired epitope to the target by designing PCR primers encoding the epitope. A further breakthrough was achieved with the development of recombination-based cloning vectors (17,18), which have made it unnecessary to create a new construct each time fusion to a new tag is desired.

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