Unlocking the Protein Toolbox: How to Choose the Right Tag for Your Experiments

Protein tags have become indispensable in recombinant protein research, offering solutions to the persistent challenges of producing proteins in systems where they are not naturally expressed. Many recombinant proteins are difficult to isolate in sufficient quantity or purity, and they may misfold, aggregate, or degrade without additional support. By attaching short peptide or protein sequences, typically at the N- or C-terminus of a protein, researchers can simplify detection, streamline purification, and enhance solubility and stability. But not all tags are created equal. Because the choice of tag can significantly influence experimental outcomes, understanding the strengths and limitations of different tagging strategies is vital to selecting the one best suited for your specific application.

Common Functions of Protein Tags

Researchers can leverage different protein tags to accomplish a wide spectrum of functions. Broadly, tags fall into the following functional categories:

• Purification - Also called affinity tags, these tags facilitate the rapid isolation of recombinant proteins from complex mixtures using affinity resins or columns. Common examples include polyhistidine (His-tag), glutathione S-transferase (GST), maltose binding protein (MBP), and Strep-tag.[i]
• Detection - To enable detection of target proteins in immunochemical and immunocytochemical applications, researchers can add a known epitope tag to a recombinant protein. This is particularly useful in instances where antibodies are not available for the target protein, or for proteins with poor immunogenicity or low abundance. Epitope tags and their respective antibodies can be used in western blot, flow cytometry, immunofluorescence, and other applications. Some examples include hemagglutinin (HA), c-Myc, and V5.[ii]
• Solubility/Folding Enhancement - Solubility-enhancing tags help to counteract misfolding and aggregation of recombinant proteins by acting as highly soluble fusion partners that promote proper folding and keep the target protein in solution. While these tags are vital to working with certain proteins, they are relatively large and may need to be removed after purification if they interfere with the protein’s native function. Examples of solubility-enhancing proteins include maltose-binding protein (MBP), glutathione S-transferase (GST), and small ubiquitin-like modifiers (SUMOs).[iii]
• Stability/Half-Life Extension - Tags such as Fc-fusions or albumin-binding domains can increase the circulating half-life and reduce degradation of therapeutic proteins. These modifications not only enhance experimental reproducibility but also the expand the clinical potential of protein-based drugs.[iv]

While some tags, such as MBP or GST, can serve multiple functions, their suitability for a particular application is dependent on the protein of interest, as well as other experimental conditions and goals.

How to Choose the Right Tag

With countless protein tags available, selecting the right one can feel overwhelming, especially if you’re new to a specific technique. When choosing a protein tag, here are some important factors[v] to consider:

• Downstream application: What kind of experiments will the protein be used in? In structural studies, for example, minimal or cleavable tags might be preferable, whereas imaging studies may need fluorescent tags.
• Size of the tag: Small tags may interfere less with native protein function but offer no benefits for solubility or stability. Conversely, larger tags can enhance protein solubility or stability but can be disruptive to activity or oligomerization.
• Ease of removal: Are you aiming to ultimately cleave off the tag to achieve a tag-free end product? If so, determine whether there may be complications or limits to cleavage that could impact function.
• Expression system compatibility: Different expression hosts place different constraints on protein and tag performance. In coli, the bacterial environment generally doesn’t complicate purification but can lead to misfolding. Mammalian cells allow post-translational modifications and complex folding, so tags that enhance stability and facilitate purification tend to be more common.
• Detection requirements: How do you plan to detect or track your protein? For western blot, immunofluorescence, or immunoprecipitation, short epitope tags may be ideal due to the wide availability of reliable, high-affinity antibodies. In highly quantitative applications, engineered tags that provide stronger, cleaner signals may be preferable. Ultimately, the right choice depends on whether your priority is sensitivity, compatibility with specific assays, or the ability to multiplex tags for multiple readouts.

 Examples of Commonly Used Tags

Customizable Options for Your Experiments

Even with the wide number of protein tags available today, researchers shouldn’t have to be limited to existing off-the-shelf options. Aviva’s Protein on Demand Semi-Custom Recombinant Proteins empower researchers at every stage with the flexibility of customization without the high cost of fully custom options.

Users can modify our collection of >300,000 protein sequences with their chosen tag and select an expression system to generate customized, high-purity proteins suitable for their research goals and scale. Production quantities range from as little as 20 µg to 1+ mg. The end result: a protein tailored for success in your hands.

Explore our Protein on Demand to learn more or request a quote for your protein and tag of choice.

Supporting Successful Research

Ultimately, selecting the right protein tag is a careful balance between purpose, function, and the specific demands of your experiment. No tag is universally “best”. The right choice depends as much on practical considerations and constraints as on the scientific goals of your project. By weighing these factors thoughtfully and seeking guidance where necessary, researchers can avoid common pitfalls and set their experiments up for clear, reliable, and reproducible results.

With Protein on Demand, Aviva Systems Biology is dedicated to helping researchers design proteins that meet their experimental needs and empower impactful research. Get in touch to learn more about Protein on Demand and Aviva’s other customer-focused products and services.

References

[i] Kimple ME, Brill AL, Pasker RL. Overview of affinity tags for protein purification. Curr Protoc Protein Sci. 2013 Sep 24;73:9.9.1-9.9.23. doi: 10.1002/0471140864.ps0909s73. PMID: 24510596; PMCID: PMC4527311.
[ii] Brizzard, B. (2008). Epitope Tagging. BioTechniques, 44(5), 693–695. https://doi.org/10.2144/000112841
[iii] Costa, S., Almeida, A., Castro, A., & Domingues, L. (2014). Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: The novel Fh8 system. Frontiers in Microbiology, 5, 77021. https://doi.org/10.3389/fmicb.2014.00063
[iv] Koehler, Michael F T et al. “Albumin affinity tags increase peptide half-life in vivo.” Bioorganic & Medicinal Chemistry Letters vol. 12,20 (2002): 2883-6. doi:10.1016/s0960-894x(02)00610-8
[v]Terpe, K., Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol. (2003) 60:523-533 DOI: 10.1007/s00253-002-1158-6