Protein Tag

Protein Tag Definition

What is Protein Tag

Protein tag refers to the sub-domain or the peptide sequence of a fusion protein (see What is Fusion Protein) that is linked to the interest protein. There are many kinds of protein tags which are widely used in recombinant protein production. Protein tags are useful and convenient tool for improving solubility of recombinant proteins, streamlining protein purification, and allowing an easy way to track proteins during protein expression and purification.

Usage of Protein Tags

A number of protein tags and peptides have been used for recombinant protein purification and the tagged protein can be easily and conveniently purified by affinity chromatography. These fusion tags contain staphylococcus protein A, glutathione-S-transferase, maltose-binding protein, cellulose-binding protein, chitin-binding domain, thioredoxin, strepavidin, RNaseI, polyhistidine, human growth hormone, ubiquitin, and antibody epitopes.

Another main application of fusion tags linked with the recombinant proteins is to enhance their solubility where Maltose binding protein, NusA, Glutathione-S-transferase, TrxA, Fh8, SUMO, Z-tag and Halo tag are used.

Fusion tags also can be used as reporters to track the expression level of the specific protein that the researchers wish to study, where β-galactosidase, luciferase, and fluorescent proteins are included. The most common used fluorescent proteins include green fluorescent protein (GFP), orange fluorescent protein (OFP), red fluorescent protein (RFP) and yellow fluorescent protein (YFP).

Review of Fusion Protein Tags

Fusion Tags Used in Affinity Chromatography

Fusion agents are widely used as affinity tags. The interaction with the specific ligand and elution under special conditions result in a highly efficient one-stage purification some fusion tags allow the refolding to be realized directly on the affinity column. A brief list of affinity tags characteristics are as shown below and see the details, click the Affinity Tags.

Characteristics of Main Affinity Tags

Fusion tag Amino Acid Sequence Matrix Elution Conditions Notes
Polyhistidine tag(Poly-His) nH (n = 2-12, usually 6) metal ions (Ni2+, Co2+, Сu2+, Zn2+, Fe2+) 20-250 mM imidazole or low pH  
Polyarginine tag (Poly-Arg) nR (n = 5-6, usually 5) cation-exchange resin NaCl to 400 mM at pH > 8.0  
FLAG DYKDDDDK (8) anti-FLAG mAb pH 3.0 or 2-5 mM EDTA site of hydrolysis with enteropeptidase
Streptavidin-binding protein(SBR) MDEKTTGWRGGHVVEGLA
Streptavidin-binding tag(Strep-tag) WRHPQFGG(8) streptavidin 2 mM biotin, mild conditions  
Modified streptavidin binding tag(Strep-tag II) WSHPQFEK (8) modified streptavidin(Strep-Tactin) 2.5 mM desthiobiotin  
Doubled streptavidin binding tag(Twin-Strep-tag) SAWSHPQFEKGGGSGGG
modified streptavidin (Strep-Tactin) 2.5 mM desthiobiotin  
Calmodulin-binding protein (CBP) KRRWKKNFIAVSAANRFKKI
calmodulin EGTA or EGTA with 1 M NaCl  
chitin bound with intein: 30-50 mM dithiothreitol, 2-mercaptoethanol used only in tan dem with intein
Maltose-binding tag (MBP) 396 transversely-linked amylose 10 mM maltose increases protein solubility
Glutathione S-trans-ferase (GST) 211 glutathione 5-10 mM reduced glutathione increases protein solubility
Cellulose-binding tag 27-189 cellulose family I: guanidine HCl or urea > 4 M  
Natural histidine affinity tag (HAT) KDHLIHNVHKEFHAHAHNK (19) Со2+ 150 mM imidazole or low pH  
c-myc EQKLISEEDL (11) anti-Myc epitope of mAb low pH  
S KETAAAKFERQHMDS(15) S-fragment of RNase A 3 M guanidine thiocyanate; 0.2 M citrate at pH 2.0; 3 M magnesium chloride  
Biotin acceptor protein (BAP) GLNDIFEAQKIEWHE(15) avidin, modified streptavidin 2 mM D-biotin  
Terbium-binding protein DDDWDDDWDDDWDDD (15) metal ions Tb3+ 100 mM EDTA luminescent tag
Hemagglutinin tag (HA) (YP)YDVPDYA (7-9) antiHA epitope of mAb 3 M NaSCN or 50 mM NaOH at low pH  

Fusion Tags Used for Enhancement the Solubility of Recombinant Proteins

Many recombinant proteins are produced with poor solubility where fusion tags are used to increase the solubility. Some of them also have the function of affinity label that makes possible good purification such as Maltose-binding protein (MBP) and Glutathione-S-transferase (GST). Sometimes two and more tags are used in a different case which is called tandems.

NusA Tag. NusA protein, a sequence of 495 amino acids, is typically used to increase expression and solubility when joined to N-term or C-term. It can influence the activity of RNA polymerase and cause stops of this enzyme on some RNA motifs. It also can be used as a component of an antiterminator complex linked with other proteins such as 6His.

TrxA. TrxA is a thioredoxin of Escherichia coli. With a 12 kDa molecular weight, TrxA can be located on either the N or C-end of the desired protein where N-end is more efficient. Like thioredoxin, TrxA is unable to bind with sorbents and it can be used together with 6His or some other partner protein tags of affinity features in protein purification.

Fh8. Fh8 protein is a small molecule of only 8 kDa produced by the liver fluke Fasciola hepatica. The protein is resistant to high temperature and retains its secondary structure on heating even to 74°C. Fh8 can be used for production of proteins with high molecular weight.

SUMO. SUMO is short for Small Ubiquitin-like Modifier, the 100 amino acid sequence, which is necessary for regulation of protein transport and is important for controlling transcription for eukaryotic cells. Sumo tag is most frequently used as N-end fusion sequence in yeast to increase the expression and solubility of the desired recombinant protein. (See SUMO Tag and SUMO Tag Purification)

Z-tag. Produced based on staphylococcus protein A, Z-tag is capable of binding with the Fc-fragment of antibodies. Z-tag is also used to increase the solubility in recombinant protein expression.

Halo. Halo is a 33-kDa protein based on a modified haloalkane dehalogenase (DhaA) found in bacteria Rhodococcus. Halo tag is also used to increase the solubility of the fused recombinant protein. Moreover, Halo can be used for protein purification when it is linked with sorbents as Halo tag can form strong covalent bonds with chloroalkanes.


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.
Kimple ME, et al. (2013) Overview of affinity tags for protein purification. Curr Protoc Protein Sci 73: 9.9.1-9.9.23.
Amarasinghe C, et al. (2015) The use of affinity tags to overcome obstacles in recombinant protein expression and purification. Protein Pept Lett 22(10): 885-892.

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