Epitope tags can be broadly categorized as peptide tags, protein tags, and fluorescent protein reporters. The following are some of the most widely used examples:
Peptide tags
A His-tag, also known as a polyhistidine tag, comprises a series of 6 to 10 histidine residues. The 6 residue format (6-His), which has a molecular weight of just 0.8 kDa, is used most often. An advantage of His-tags is that they allow for affinity purification using inexpensive and readily available nickel or cobalt resins.
The S-tag consists of the N-terminal 15 amino acid residues of pancreatic RNase A (KETAAAKFERQHMDS) and has a molecular weight of 1.8 kDa. It binds with high affinity to the S-protein, which comprises residues 21-124 of the same RNase enzyme.
The DYKDDDDK-tag, also known as the FLAG®-tag, is a hydrophilic epitope tag with a molecular weight of 1 kDa. It can be added singly, in tandem, or in triplicate, with the most commonly used triplicate motif being DYKDHDG-DYKDHDI-DYKDDDDK. The DYKDDDDK-tag is easily removed with enterokinase, which cleaves after the lysine (DDDDK^X).
The Myc tag is a 10 amino acid peptide (EQKLISEEDL) with a molecular weight of 1.2kDa that is derived from the Myc proto-oncogene. It is frequently used for immunoassays such as western blot and flow cytometry, but less often for purification since elution requires low pH, which risks damaging the tagged protein.
Protein tags
- Glutathione S-Transferase (GST)
GST is a 26 kDa protein, derived from the parasite Schistosoma japonicum, that binds with strong affinity to its substrate, glutathione. It is useful for increasing the solubility of recombinant proteins, especially those expressed in bacteria. However, the large size of GST can interfere with some protein functions and often necessitates its removal by protease cleavage.
- Maltose-Binding Protein (MBP)
MBP is a 43 kDa protein, encoded by the malE gene of Escherichia coli, that allows for affinity purification using amylose resins. It is one of the most common crystallization chaperones and, like GST, can improve the solubility of recombinant proteins expressed in bacteria.
- Small Ubiquitin-like Modifier (SUMO)
SUMO is a 12 kDa protein that regulates protein function by serving as a post-translational modification. It has been shown to improve the stability and solubility of recombinant proteins and is easily removed using SUMO-specific proteases.
Fluorescent protein reporters
- Green Fluorescent Protein (GFP)
Originally derived from the jellyfish Aequorea victoria, GFP is one of the longest established fluorescent proteins for scientific research. It has a molecular weight of 27 kDa and is widely used for immunofluorescence, flow cytometry, and fluorescence resonance energy transfer (FRET), as well as for live-cell imaging studies.
DsRed, a 30 kDa protein isolated from Discosoma sp., was the first red fluorescent protein (RFP) to be discovered. A main advantage of DsRed, and RFPs generally, is that the red-shifted wavelength spectrum avoids damaging cells and tissues by excitation light, as well as is distinct from autofluorescence.
mCherry is a second-generation RFP that was developed via the directed mutagenesis of DsRed. It has a molecular weight of 27 kDa and benefits from good photostability, rapid maturation (assembly), and low acid sensitivity.
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