The fusion of a small protein or peptide (tag) to the protein of interest is a commonly used method to aid purification of recombinant proteins. Fusion tags can improve protein expression, stability, resistance to proteolytic degradation and solubility. A wide range of fusion tags are available from small peptides to relatively large proteins, each with its own unique characteristics. Many solubility tags are engineered for use in bacterial expression systems to overcome poor protein solubility.

Fusion Tag Function Size (kDa) Description
Polyhistidine (e.g. 6xHis, 10xHis) Affinity 1-2 The most commonly used affinity tag, binds to metal ions
Strep-tag II Affinity 1 High affinity for engineered streptavidin
Thioredoxin (Trx) Solubility 12 Aids in refolding proteins that require a reducing environment
Small Ubiquitin-like Modifier (SUMO) Solubility 12 Contains a native cleavage sequence enabling tag removal with SUMO protease
Glutathione S-transferase (GST) Solubility, affinity 26 High affinity for glutathione, often needs to be removed due to large size
Maltose Binding Protein (MBP) Solubility, affinity 41 Binds to maltose, often needs to be removed due to large size
Fusion Tag Orientation

In any fusion tag system, the sequence encoding the tag is placed directly upstream or downstream of the recombinant gene sequence. A fusion tag that increases solubility or expression is most beneficial if placed at the N-terminus, acting as a positive translation initiator. Fusion tags primarily used for affinity purposes can be useful on the N- or C-terminus. If tag removal is desired, the sequence is often placed at the N-terminus to minimise the number of leftover residues following cleavage.

Combinatorial Fusion Tags

A combination of fusion tags can be used to maximise their functionality. A popular method is to utilise a solubility tag (e.g. GST or MBP) and an affinity tag (e.g. polyhistidine). This combination promotes soluble protein production and provides multiple options for affinity purification.

Fluorescent Proteins

Bioluminescent proteins are used in a wide range of biological applications from ultra-sensitive assay detection to in vivo imaging of cellular processes. The current range of bioluminescent proteins are variations of the wild-type Green Fluorescent Protein (GFP) derived from the jellyfish Aequorea victoria. Genetic variants featuring fluorescence emission spectral profiles spanning the blue, cyan, red, and yellow regions of the visible spectrum were developed by engineering specific mutations into GFP. These probes are used in a wide range of applications such as:

  • Fluorescent Resonance Energy Transfer (FRET)
  • Fluorescent Activated Cell Sorting (FACS)
  • Photoactivated Localisation Microscopy (PALM)
  • Fluorescence Recovery After Photobleaching (FRAP)
  • Confocal Microscopy
Tag removal

In many cases it is desirable to remove fusion tags during purification to restore native protein structure. Removal of the tag is achieved by including a cleavage site between the fusion tag and the gene sequence. Commonly used cleavage proteases include:

Protease Type Recognition Site
Human Rhinovirus 3C (HRV3C) Cysteine LEVLFQ/GP
Tobacco Etch Virus Protease (TEVp) Cysteine ENLYFQ/G
Ubiquitin-like Specific Protease (SUMOp) Cysteine Recognises tertiary structure
Thrombin Serine LVPA/GS

A number of challenges can be encountered during tag removal, including:

  • Incomplete cleavage
  • Difficulty in separating the protease and tag from the native protein
  • Loss of protein from the cleavage process
  • Loss of solubility following cleavage