Pairing your Protein with a Purification Tag

With the introduction of affinity tagging of proteins, protein purification was dramatically simplified; generic protocols could now be used, which enabled much more efficient and easy protein purification. This led to tags that do more than just purify protein, such as improving the solubility or stability.

In this post we’ll look at the characteristics, pros and cons of the most common tags used in protein purification today.

The “classic” His-tag

The histidine tag is by far the most commonly used tag for protein purification today. The reason for this is simple – it is so small that it is unlikely that it will interfere with the structure or function of the protein. This means that you don’t necessarily have to remove it before using the purified protein (one example when the his-tag often needs to be removed is for structure determination using X-ray crystallography). Another great benefit of using this tag is that purification is quite straightforward, and there is a great selection of ready-to-use purification products in a multitude of formats available to choose from. Different chromatography media are available that will provide different trade-offs between recovery, capacity and purity.

Adding histidine tag means that you typically add 4-6, sometimes up to 10, histidine residues to either the N- or the C-terminal of your protein. The aromatic group of the histidine residues bind to chelated di-valent metal ions. Nickel is the most commonly used, but Cobalt, Zinc and Copper can also be used. Zinc is the best choice for the environment. Regardless of the ion, imidazole is always used for the elution.

The main drawback of this tag is that it often requires some optimization of the imidazole concentration in your sample and in the buffers for column equilibration and wash in order to minimize binding of other host cell proteins with high histidine content.

Strep-tag™ II

Strep-tag II is a peptide tag that binds very specifically to Streptactin™, which is a modified version of streptavidin. Being small, it shares the benefits of the His-tag, and adds significant improvement in the purity you can expect.

In addition to the chromatography media being more expensive and having much lower binding capacity than media for purification of his-tagged proteins, the agent used for elution, desthiobiotin, is more expensive than imidazole.

GST for purity and solubility

Another very common tag is the enzyme Glutathione-S-Transferase (GST). It binds very specifically to glutathione immobilized on chromatography media, and therefore often gives very high purity. Another benefit is that it can also increase the solubility of the protein it is fused to. However, being big (26 kDa) it often needs to be cleaved off in order to eliminate interference with structure and function of your protein.

While there are chromatography media with high binding capacity, the kinetics of the binding is slow. The latter means that sample loading needs to be done at low flow-rates and therefore will take longer compared to e.g. a his-tagged protein.

MBP tag

Maltose Binding Protein (MBP) is another protein tag that can be used for purification and as an alternative to GST. Whilst providing the same benefit of high specificity and ability to improve solubility of your protein, it is larger than GST, so typically requires removal prior to using your protein. Lower binding capacity compared to GST and a more limited number of purification products available make this tag a second choice if the GST-tag for some reason does not work.

FLAG™ tag

If none of the to the tags discussed above work, the FLAG peptide-tag is a small tag that binds very specifically to a specific antibody currently only available on one type of chromatography media. In addition to the high specificity and thereby purity that can be expected, another benefit of this tag is that it is small, and therefore is unlikely to interfere with the function of the protein it is fused to.

The main drawback is that the affinity media is based on an immobilized antibody, and therefore has a limited binding capacity, resulting in either larger column sizes or smaller amounts purified per batch. The chromatography media also comes at a higher cost than alternative affinity media.

Key + low +++ high

Conclusion

There are many other tags that you can use but we hope this overview will give you ideas for what you should consider when choosing a tag for your protein. The key is to know your protein and the tag’s characteristics for a good choice and purification.

For more information, tips and ideas on tagged proteins, view our webinar with Professor Richard Burgess, Editor in Chief of the journal Protein Expression and Purification, and also check out this excellent collection of papers collated by Dr Richard R. Burgess which is an excellent review and includes research articles illustrating many of the purification tags in current use, including detailed experimental descriptions, example protocols, strategies and best practices for using tags.
What experiences to do you have from such tags? Have you seen other benefits or drawbacks when using the tags described here? Please use the comment field below to share your thoughts.