You have identified and cloned your protein of interest, but you want to explore its function. A protein fusion tag might help with your investigation. However, choosing a tag for your protein depends on what experiments you are planning. Do you want to purify the protein? Would you like to identify interacting proteins by performing pull-down assays? Are you interested in examining the endogenous biology of the protein? Here we cover the advantages and disadvantages of some protein tags to help you select the one that best suits your needs.

Affinity Tags

The most commonly used protein tags fall under the category of affinity tags. This means that the tag binds to another molecule or metal ion, making it easy to purify or pull down your protein of interest. In all cases, the tag will be fused to your protein of interest at either the amino (N) or carboxy (C) terminus by cloning into an expression vector. This protein fusion can then be expressed in cells or cell-free systems, depending on the promoter the vector contains.

Tags with Noncovalent Binding

Polyhistidine or 6XHis tag is a six histidine tag frequently used for creating fusion proteins. Its small size tends not to disrupt protein structure, so you can study the protein in other assays. The polyhistidine tag binds to metal ions like Ni²⁺ and Zn²⁺, making it easy to purify the fusion protein when a crude lysate is passed over a resin that contains nickel, binding the polyhistidine-tagged proteins. Proteins fused with polyhistidine are best expressed in bacterial cells where there is little background binding from other proteins. If expressed in insect or mammalian cells, more stringent washing of the metal resin is needed to remove other proteins that have polyhistines in their sequence.

Glutathione-S-Transferase (GST) is another common affinity protein tag that is used for proteins expressed in E. coli to purify the tagged proteins from bacterial lysates. At 26kDa, GST is larger than polyhistidine and a eukaryotic protein, making GST problematic to use with mammalian or insect cells. While the larger tag can interfere with protein function, GST can make fusion proteins more soluble and express at greater levels in E. coli, a benefit for purifying more protein at one time. GST binds to glutathione, and using beads coated in glutathione can capture and purify the fusion protein.

Tags with Covalent Binding

Despite the affinity the polyhistidine tag has for metal ions and GST has for glutathione, the interactions are reversible and can be disrupted when applying stringent washing. A covalent bond offers a method to not only capture a tagged protein but its strength means you can eliminate background binding with stringent washes. HaloTag® Technology is based on the covalent interaction between the 34kDa HaloTag® protein and its ligands. Once you create a N- or C-terminal fusion protein with HaloTag, you can purify the tagged protein, track its movements in the cell, study protein:protein interactions and more. This affinity tag works in both E. coli and mammalian cells. Expressing a tagged protein in mammalian cells means the protein will contain post-translational modifications, more accurately reflecting its cellular function. In addition, once you purify the protein, HaloTag can be removed by TEV protease cleavage, giving you purified, untagged protein for your analyses.

Bioluminescent Reporter Tags

There are bioluminescent tags are available in two options: full-length reporter gene or a tiny peptide (11 amino acids) that generates light in the presence of a second complementary protein. Similar to affinity tags, bioluminescent tags are fused to your protein of interest at the N or C terminus. However, you may be able to skip cloning step and add the tiny HiBiT peptide tag using CRISPR-Cas9 gene editing. This means adding the tag to the endogenous gene and examining your protein of interest in the context of the cell. Most tagging methods use exogeneous expression of the tagged protein, which means the tagged protein is expressed from a plasmid at much greater levels than that found normally in cells. However, this greater expression level does not accurately reflect the amount of protein in the cell and can distort the biology being studied.

In addition, using a bioluminescent reporter like NanoLuc® luciferase, you can detect the light from the protein in the cell, tracking its movements and any translocations (e.g., if a virus containing a bioluminescent reporter successfully infects a cell). The brightness of NanoLuc® luciferase and the HiBiT tag can be measured using both lytic and live-cell methods, offering sensitive detection of the tagged proteins.

Epitope Tags

If you have a newly discovered protein but want to use it in various immunoassays like Western blotting, immunoprecipitation, immunohistochemistry, immunocytochemistry or flow cytometry, an epitope tag might benefit you. An epitope tag is typically a short peptide that can be added to the protein coding sequence via PCR or using a cloning vector that contains the epitope coding region. For novel or poorly immunogenic proteins, an epitope tag provides a way to use commercially available antibodies for immunoassays. Commonly used epitope tags include hemagglutinin (HA), c-Myc and FLAG, and are added to the amino (N) or carboxy (C) terminus of a protein. The HiBiT peptide tag functions as an epitope tag when using it with the Anti-HiBiT Monoclonal Antibody, and can be used for FACS analysis as well as Western blotting and immunoprecipitation. In addition, when the HiBiT tag is added to an endogenous protein using CRISPR/Cas9 knock-in, you can confirm subcellular localization.

However, epitope tags need to be added in frame with its fusion partner. Using epitope tags for protein purification can be problematic for both binding to a substrate and protein functionality, depending on the tag used. Because some tags are derived from mammals, cross-reactivity is a potential problem.

Summary

Depending on your experimental goals, there a protein fusion tag that will help with your experiments. Whether you are seeking an affinity tag or bioluminescent reporter, you will need to analyze the benefits of each peptide tag to select the right one for your goals.

Resources

Want to learn more about protein tagging? This article has more information on the protein tags described in this post, including all their potential applications.

Note: This post was updated April 2023.

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Sara Klink

Technical Writer at Promega Corporation

Sara is a native Wisconsinite who grew up on a fifth-generation dairy farm and decided she wanted to be a scientist at age 12. She was educated at the University of Wisconsin—Parkside, where she earned a B.S. in Biology and a Master’s degree in Molecular Biology before earning her second Master’s degree in Oncology at the University of Wisconsin—Madison. She has worked for Promega Corporation for more than 15 years, first as a Technical Services Scientist, currently as a Technical Writer. Sara enjoys talking about her flock of entertaining chickens and tries not to be too ambitious when planning her spring garden.

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