Epitope Tagging with a Focus on Polyhistidine Tags

Protein purification is one of the cornerstones of biological studies. Identifying protein function typically first requires intense protein-purification protocols. One way to purify a protein is through the use of epitope tagging. Using recombinant DNA cloning methods, epitope-tag sequences can be added to a protein of interest. Epitope tagging is accomplished by fusion of the target protein with the tag of choice. This is performed by inserting the target gene into a host cell-specific expression vector that also encodes the epitope tag. Expression vectors have been developed for a variety of host cell types, including Escherichia coli, yeast, insect and mammalian cells.

Here we provide an overview of the more commonly used options and also discuss some tips and considerations when selecting an epitope tag to use. The goal is to achieve the best expression and yields of your target protein.

Many choices

Many different affinity tags are currently available, including glutathione S-transferase (GST), polyhistidine (His6) and the more common epitope tags human influenza hemagglutinin (HA), protein C heavy chain (C-Tag), c-Myc and FLAG octapeptide. The FLAG system, which utilizes a sequence motif of DYKDDDDK, is commonly used to detect recombinant proteins expressed in mammalian host cells. The FLAG tagging system includes the anti-FLAG M1 monoclonal antibody with calcium-dependent binding. FLAG-tagged proteins can be eluted from the M1 antibody with EDTA.

Often, the epitope tag is not necessary post-purification—nor desirable on the recombinant protein product. So in many cases, the recognition sequence for a protease is attached after the coding sequence, to enable release of the epitope tag. Specifically, tags can be cleaved from the protein of interest using widely available proteases, such as enterokinase, tobacco etch virus protease and the rhinoviral 3C protease; these are available from various commercial sources.

Overcoming the bottlenecks

One of the most commonly used tags to purify and detect recombinantly expressed proteins is the polyhistidine (His6) tag. His6 tagging relies on the principles of affinity metal chelate chromatography. In other words, it uses the specific and reversible binding of a His-tagged protein to a matrix-bound ligand, which in this case is nickel-NTA. Depending on the downstream application, affinity purification might be the only chromatographic step required to achieve adequate purity. This particular type of affinity chromatography is believed to result from coordination chemistry between a nitrogen on the imidazole moiety of polyhistidine with a vacant coordination site on the metal. Once bound to the metal, the protein of interest is immobilized to the column or beads through complex formation with a chelate that is covalently attached to the column or beads.

Polyhistidine tags offer several advantages for protein purification. The small size of the tag is one such advantage, as it creates a tag that is less immunogenic than other larger tags. Also, in this case, the tag usually does not need to be removed for downstream applications post-purification. Furthermore, the polyhistidine tag is available from a large number of commercial expression vectors. Finally, placement of this small polyhistidine tag at either an N- or a C-terminus of a protein of interest does not impact expression, in most cases.

Protein purification of His-tagged proteins has traditionally been performed using nickel-NTA columns. But this system is fraught with technical bottlenecks, including long equilibration and binding times as well as slow diffusion of large molecules through the resin bed. To circumvent these bottlenecks, Clontech, a Takara Bio Company, has created the Capturem™ His-Tagged Purification Mini and Maxi Kits. These kits make use of next-generation membrane technology that enables the researcher to attain high-purity, high-throughput purification with a five-minute, room-temperature protocol. Using spin columns assembled with novel nylon-based membranes specifically modified to have a much higher protein-binding capacity than conventional membrane pores, Capturem™ minipreps accommodate loading of up to 750 µl of lysate from mammalian or bacterial cells, and they yield more than 90% of the His-tagged protein present in the culture (approximately 100 µg per column). “This system also greatly reduces the possibility of protein degradation and loss of activity, partly due to its short residence time, during which the protein is bound to the membrane,” says Keren Drori, product manager at Clontech. “This, combined with the ability to elute in a small volume and preload your buffer in the collection tubes, protects your protein from degradation. Because of this technology, researchers were able to purify an active, hydrophobic ion channel protein in just five minutes.” Researchers can now screen hundreds of clones quickly and efficiently, in search of the clone with the best expression. The rapid purification steps enable researchers to test the expression of their protein before scaling up, saving both time and money.

“Researchers can take advantage of flexibility often lacking with traditional nickel-NTA resins,” says Drori. For example, the Clontech kits are compatible with the presence of common additives (EDTA, β-mercaptoethanol, TCEP, dithiothreitol, glycerol and more) and allow scientists to purify the protein while using protease inhibitors to protect the protein from degradation.

Other pluses and minuses

Each of the epitope-tagging systems has its own benefits and limitations. For example, GST, maltose-binding protein and larger tags can confer solubility and stability when fused to a smaller protein of interest. Among the benefits of other more established systems, “epitope tags such as HA, C-Tag, Myc and FLAG are small and generally do not change the structure or function of the protein of interest,” says Peter Bell, senior director, research and development, protein biology, at Thermo-FisherScientific. On the other hand, “epitope-tag purification, such as with HA, C-Tag, Myc and FLAG, require more expensive media due to the fact that each of these require an immobilized antibody directed against the short epitope-tag sequence,” he says.

Epitope tags are powerful tools for protein characterization. There are many options to consider in making a selection that will work best with your protein of interest. This will require some protocol optimization as well as trial-and-error testing, but you will soon be on your way to purifying and characterizing your proteins.

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