Fusion tag

In genetic engineering, a fusion tag is a protein added to the N- or C-terminus of a specific protein of interest (called the 'target protein') in order to facilitate one or more of the following:

  • Solubilisation of the target protein, by fusing the N-terminal of the target protein with the C-terminal of a soluble compound

  • Detection of the target protein, by fusing either terminus of the target protein with an epitope (antigen-like) tag for hybridisation with antibodies, or a biophysical indicator such as a fluorescent protein

  • Purification of the target protein, by fusing peptides to either terminus of the target protein that bind specifically to affinity resins

  • Localisation of the target protein (to a particular cellular destination), by fusing a tag, usually on the N-terminus of the target protein, which signals cellular transport to. a particular organelle

  • Enhanced expression of the target protein, by fusing the C-terminus of an already highly expressed protein to the N-terminus of the target protein

Fusion tags are engineered in the form of recombinant DNA, by artificial insertion of the additional gene sequences that will create the recombinant protein. The 'fusion gene' must be continuous with the gene coding for the target protein in terms of open reading frame; i.e. any stop codons between the two fusion partners must be omitted.

In addition to the list above, fusion tags can assist in establishing the correct folding pattern of the target protein. However, detrimental effects of fusion tagging have also been reported, including but not limited to: protein misfolding, biological inactivity of the protein, poor yield and even toxicity. For this reason it is important that tags are removed soon after their use in expression. The tags should thus be customised with a view to how they will eventually be removed by proteolytic cleavage. Ultimately it is desirable to have only the native protein isolated and not any extraneous amino acid sequences.

Fusion tags come broadly in two categories: affinity tags, that aid in purification but do not enhance solubility, and solubility-enhancing tags that do enhance solubility and expression, and may or may not contribute to purification.

Affinity tags are the most commonly used tag for aiding in protein purification. They can be defined as exogenous amino acid sequences that bind with high affinity to a chemical ligand or an antibody. Most affinity tags are short peptide sequences that either bind to a ligand linked to a solid support (like the His tag) or contain an epitope recognised by immobilised antibodies (like the FLAG or Myc tags). The high affinity of these tags for their ligands and the availability of well developed immobilised supports for capturing the fusion proteins allow the target protein to be purified to a very high degree. Because of their small size, these affinity tags can be added at either end of the protein or in an internal region of the protein, providing it is exposed to the surface. However, these tags generally do not increase the expression of the fusion proteins or enhance their solubility, and therefore are of little use in purifying hard-to-express proteins. His-tags are the most widely used affinity tags, coming in either His6, His8 or His10 forms, depending on the length of the histidine repeat. The purification of his-tagged proteins is based on the use of a chelated metal ion as an affinity ligand (such as Zn2+); one commonly used ion is the immobilised nickel-nitrilotriacetic acid chelate [Ni–NTA], which is bound by the imidazole side chain of histidine. Removal of tags at the end of the purification process is essential and this is done using an immobilised subtilisin protease enzyme which carries out both affinity binding and tag cleavage (the latter only upon application of an elution buffer).

Solubility-enhancing tags are generally large peptides or proteins that increase the expression and solubility of fusion proteins. Fusion tags like GST and MBP also act as affinity tags and as a result, they are very popular for protein purification. Other fusion tags like NusA, thioredoxin (TRX), small ubiquitin-like modifier (SUMO), and ubiquitin (Ub), on the other hand, require additional affinity tags for use in protein purification.

Proteases that may typically be used in tag cleavage include enterokinase (the trypsin-activating enzyme found in the gut); factor Xa from the blood-clotting cascade and tobacco etch virus (TEV) protease.

Because every protein is unique, no single tag or cleavage method will answer every need. For proteins that express well, the simplest affinity tags may be sufficient (e.g., His6, myc). For harder to express proteins, fusion partners that enhance folding and solubility are preferable (e.g., MBP, SUMO). Tag removal then adds another layer of complexity. When considering which tag to use, key questions should be asked. For example, can your application tolerate retention of the tag, one or more amino acids remaining at the cleavage site, or must tag removal leave no trace? Such questions are usually answered experimentally, but with the availability of solubility-enhancing tags paired with highly specific proteases that cleanly remove the tag, these questions may be moot.