The Protein Man's Blog | A Discussion of Protein Research

8 Protein Tags Explained

Posted by The Protein Man on Jun 10, 2014 10:00:00 AM
The Protein Man
Basically, protein tags are peptide sequences that are attached to proteins to facilitate easy detection and purification of expressed proteins. In addition, they can also be used to identify potential binding partners for your protein of interest.

Protein Tags

At present, there is a wide variety of protein tags that can be used to suit your purpose. These include affinity tags, epitope tags and fluorescent tags.

Affinity Tags
These tags allow you to purify proteins from crude cell lysates, even if you don't have any prior knowledge of their biochemical properties. Affinity tagged proteins can be purified by using a specifc affinity resin while detection can be facilitated by using commercially available antibodies. Some of the most commonly used affinity tags include the following:

  • Glutathione-S transferase (GST): GST can be used to purify a protein by inserting its coding sequence next to your protein of interest. They will then be expressed as a fusion protein after transcription and translation. Since GST has a strong binding affinity for GSH, beads coated with the compound can be added to the mixture to isolate your protein of interest from the rest of the solution. This technique can be used to explain direct protein-protein interactions but since your protein will be attached to GST, its native state will definitely be altered. One downside of the GST tag is that it is a 30kDa protein and its relatively large size makes the purification difficult. In addition, it also increases the risk of distorting the natural conformation of the proteins and makes them inappropriate for structural studies. To prevent these things from happening, consider using a specific protease to cleave the GST group from the recombinant protein after purification.

  • Poly-Histidine tag (His): This tag comprises of 6-8 histidine residues. The relatively small size of His tags makes its integration into expression vectors extremely easy. His-tagged proteins are purified using immobilized nickel, cobalt or zinc ions, and eluted using EDTA or imidazole. Since these tags do not form secondary structures to bind their substrate, you can easily purify your protein of interest even under denaturing conditions. During purification, ensure that EDTA and other metal chelators are avoided. EDTA is often present in commercial protease inhibitor cocktails.

  • Calmodulin Binding Protein (CBP): The relatively small size of CBP (4 kDa) makes it ideal for purifying delicate proteins under mild conditions. The tag binds to a calmodulin resin and the proteins can be eluted with a neutral buffer containing low concentrations of EGTA, a calcium chelator.

  • Maltose-binding protein (MBP): These tags bind to amylase agarose and are commonly used to increase the solubility of fusion proteins.

Epitope Tags
These tags are typically smaller than affinity tags and are readily recognized by antibodies. Due to their relatively small size, they have extremely little or no effect on the structure of the resulting fusion protein. Epitope tags are ideal for several downstream applications which include western blotting, co-immunoprecipitation and immunofluorescence experiments. Some of the most popular epitope tags include the following:

  • Myc tag – This tag is a short peptide sequence (EQKLISEEDL ) derived from the c-myc gene product and recognized by numerous commercial antibodies. It can be added to a protein using recombinant DNA technology and may be used for affinity chromatography and for isolating protein complexes with multiple subunits.
  • Human influenza hemagglutinin (HA) tag – The HA tag is a peptide sequence (YPYDVPDYA) derived from the surface glycoprotein that facilitates the ability of the influenza virus to infect its host and is recognized by numerous commercial antibodies. It is extensively used as a general epitope tag in expression vectors and is useful in facilitating the detection, isolation, and purification of your protein of interest. 
  • FLAG tag – Like the Myc tag, the FLAG tag is a popular short peptide tag (DYKDDDDK) used in recombinant DNA technology and can be used for affinity chromatography and for isolating protein complexes with multiple subunits. It is recognized by numerous commercial antibodies, can be fused to the C-terminus or the N-terminus of a protein and can also be used with other affinity tags. The FLAG tag is more hydrophilic as compared to other tags in its class so they do not denature or inactivate the proteins to which they are attached.

Fluorescent Tags
Due to their non-toxic nature, these tags can be used to detect tagged proteins in both live and fixed cells. Green fluorescent protein (GFP) is one of the most widely used protein tags under this category. GFP is a protein isolated from the jellyfish Aequorea victoria that exhibits bright green fluorescence that does not fade easily when exposed to blue or ultraviolet (UV) light.

By using GFP, you can determine whether a particular promoter was activated without going through the rigorous process of measuring mRNA levels. You can also use it to observe a particular protein as it performs its role within the cell. This particular tag is remarkably stable and can function when added to either end of a protein of interest.

Topics: Protein Purification

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