Protein tags are convenient for improving solubility of recombinant proteins, streamlining protein purification, and allowing an easy way to track proteins during protein expression and purification. Perhaps the most common application of protein tags involves the addition of a purification 'tag', also called affinity tag, which provides a standardized method to purify the fused recombinant protein.
What is affinity tag? Affinity tag refers to a short peptide added to either the N- or C-end of a recombinant protein to facilitate purification of the expressed protein and the affinity tag sequence usually contains from several to hundreds of amino acids. Many tags can also provide additional functions unrelated to purification, such as facilitating detection of the target protein or improving the solubility of the target recombinant protein.
Affinity fusion tags are widely used in purification of recombinant protein.
Different affinity tags have their specific application in recombinant protein purification depend on the properties of the target protein, the elution, the expression systems and the specific conditions of affinity column.
After the expression of the tagged fusion protein, the sample is added to the purification column where the affinity tag can be adopted together with the target protein. A selective interaction with the specific affinity tag and elution under special conditions result in a highly efficient one-stage purification of the desired product.
In some cases, it is necessary to restore the biological activity and structural conformation of the recombinant protein, some fusion tags that allowing the refolding of the desired protein are required. Moreover, application of affinity tags can also lower material and time expenditures for obtaining the desired recombinant protein.
Affinity tags are highly efficient tools for protein detection, characterization, and purification. Described below are numerous affinity tags commonly used in recombinant protein production, as well as relevant caveats.
Polyhistidine tag, or nHis for short, refers to a polypeptide consisting of several histidine residues that can be located on either the N or C-end of a recombinant protein. As one of the most frequently used sequences, nHis is a small sequence that not charging the protein and not influencing the protein transfer and its folding within the cell. There are some highly specific commercial monoclonal antibodies to polyhistidine tag. In addition, Polyhistidine tag does not affect the structure and function of the purified protein.
The polyhistidine system of purification of recombinant proteins is successfully used in various expression systems, including bacterial, yeast, plant cell and mammalian cells systems. Get more details about polyhistidine tag.
Polyarginine tag (Poly-Arg or nArg tag) typically consists of five or six consecutive arginines at the C-terminal end of a recombinant protein. Poly-Arg tag is used for purification of recombinant proteins, and due to the polyarginine tag, a recombinant protein acquires a positively charged end with affinity for a negatively charged sorbent, so it may affect tertiary structure of protein and/or protein properties. In general, the polyArg tail is not the optimal purification tag in the protein-engineering field.
Recombinant protein joined by polyArg tag is purified by absorption to cation exchange resin SP-Sephadex, and eluted via a linear NaCl gradient at alkaline pH. After purification, the C-terminus of poly-Arg tagged proteins can be removed by carboxypeptidase B. What's more, polyarginine tag can immobilize targets on mica for microscopy studies. Click here to see more information about Polyarginine tag.
Glutathione-S-transferase (GST), a 26 kDa sequence of 211 amino acids, is another widely used affinity tag that increases solubility of the desired protein. GST tag has affinity for immobilized glutathione and is used for prokaryotic expression more frequently. See details of GST tagged protein purification.
FLAG tag, consisting of eight amino acids and with molecular weight of 1 kDa, is also successfully used in affinity chromatography. The small size of the tag minimizes any interfering effect. At the same time, application of FLAG tag in low pH elution may irreversibly affect protein properties.
Streptavidin-binding peptide or Streptavidin-binding protein (SBP) is 38 residues in length and can be used to immobilize fusion proteins on a streptavidin matrix. Recombinant proteins containing the SBP-tag bind to streptavidin and this property can be utilized in specific purification and detection.
The streptavidin-binding tag (Strep-tag) is a small affinity protein that is used as a sorbent. Because the affinity of biotin for streptavidin is higher than that of any of the streptavidin-binding peptides, Strep-tagged fusion proteins can be efficiently eluted by free biotin. Strep-tag is infrequently used as it is restricted by its location only on the C-end and by its relatively low affinity.
Strep-tag II, also called modified streptavidin-binding tag, is a small affinity peptide (WSHPQFEK) that was simultaneously optimized compared with Strep-tag. Strep-tag II is inert, largely resistant to cellular proteases, can be used with mild detergents. Strep-tag II works equally well on both the C- and N-ends of the recombinant protein and it is optimal for the purification of recombinant proteins under physiological conditions.
What advantages of the Strep-tag II have compared with Strep-tag? With modified streptavidin (Strep-Tactin) used as a matrix, the efficiency of binding with the sorbent could be increased and losses of the desired product decreased. So Strep-tag II can be used in purification of recombinant proteins from bacteria, yeast, mammalian, plant, and insect cells infected with baculovirus.
Twin-Strep tag, consisting of two Strep-tag®II moieties connected by a short linker, is developed to improve the binding characteristics of Strep-tag II when it is used in the protein purification from large volumes. The Twin-Strep-tag features all beneficial properties of Strep-tag II, including efficient elution under gentle competitive conditions. However, Twin-Strep-tag enables a more universal use in applications requiring stable binding because of its higher affinity.
Calmodulin binding peptide, or Calmodulin binding protein purification system utilizes a C-terminal fragment from muscle myosin light-chain kinase in order to purify proteins of interest from bacteria. This 26 amino acid fragment displays a strong affinity for the protein calmodulin. With a molecular weight of only 4 kDa, CBP tag itself is relatively small and is much less likely to affect the properties of the protein of interest.
The chitin-binding tag, or chitin-binding domain, comes from Bacillus circulans and consists of 51 amino acids. The recombinant protein binds with the sorbent (chitin immobilized on Sepharose) under physiological conditions.
CBD fusion proteins are purified by affinity chromatography on chitin resin. Commercially available vectors provide for intein-CBD expression on the N-terminus, C-terminus, or both termini of a heterologous protein of interest.
Maltose-binding protein is one of the most popular fusion tags. It is a rather large periplasmic tag with molecular weight of 43 kDa. Location of MBP on the N-end of the desired protein is more efficient than on the C-end. The desired protein fused with MBP can be expressed in either the cytoplasm or periplasmic space. Affinity chromatography on amylose is used to purify proteins fused with MBP.
Cellulose-binding tag belongs to carbohydrate binding module (CBM) family 1 and can be joined to the C- or N-end of the recombinant protein in cellulose column purification. The Cellulose-binding domain serves to promote enzymatic hydrolysis of crystalline cellulose by increasing the concentration of bacterial and fungal cellulases on cellulose surfaces.
Natural histidine affinity tag epitope is a naturally-occurring sequence of non-adjacent histidine residues that has a lower overall charge than tags with consecutive His residues.
HAT-protein fusions exhibit solubility that more closely resembles that of wild-type proteins, while still possessing strong affinity for immobilized metal ions. HAT-tagged protein is soluble, therefore it does not form aggregates and it can be eluted under mild conditions such as low imidazole concentration or neutral pH.
Wood DW (2014) New trends and affinity tag designs for recombinant protein purification. Curr Opin Struct Biol 26: 54-61.
Pina AS, et al. (2014) Affinity tags in protein purification and peptide enrichment: An overview. Methods in molecular biology (Clifton, N.J.) 1129: 147-168.
Ohba Y, et al. (2013) Fluorescent protein-based biosensors and their clinical applications. Prog Mol Biol Transl Sci 113: 313-348.