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.

When should I use a tag to purify my protein? (and when not?)

Sometimes we’re not particularly interested in purifying proteins. We just want to get it over and done with so as to get on with the experiments that will help us understand it role. This is where tagging your protein with something that adds biospecific affinity comes in handy. It allows you to simplify the purification protocol greatly, sometimes to the extent that you can use a standard protocol.

Remember though, tagging may not always be the right solution as adding a tag can introduce changes compared to the native protein, leading to undesirable effects. For example, if you are interested in drawing conclusions about function, having a tag may introduce uncertainty, or worse,  totally destroy or alters the function of your target protein. 

Also the tag itself can interfere with your ability to use the protein the way you want; important in using therapeutic proteins, where you want to be as close to the native form as possible. In most situations though tags, especially small ones like a His-tag, have no negative affects in terms of biophysical characterisation.

If you need a protein in native form

If you have decided that you need to have your protein in the native form, you can express it without a tag and go through the process of designing a protocol involving multiple chromatographic purification steps. It can be a little tedious and potentially unnecessary. Nowadays, with so many proteins already having been expressed, it is likely that you will be able to find existing scientific literature with information on your purified your protein, or an analogue.

As an alternative to expressing and purifying the native form, you can tag your protein and then remove it later. Whilst this simplifies the purification protocol, you’ll have to figure out what tag to use and how to remove it. You will also have to add the steps for cleaving the tag (manually removing it) to your protocol. It does not always have to be manual; there are systems such as AKTA pure that can automate this for you.

Removing the tag sometimes  isn’t very straightforward. It always includes using a sequence specific protease. The blood factors Thrombin & Factor Xa are most commonly used for this, but you need to make sure that their respective cleavage sequences (LVPR↓GS and IEGR↓) are not present in your target protein. Some tags, such as the Glutathione-S-Transferase (GST)-tag can be removed with a more specific proteases, such as PreScission protease (human rhinovirus 3C protease, cleavage sequence LEVLFQ↓GP), and this is extremely unlikely to effect your protein.

If you don’t need a protein in its native form

In addition to using a tag to simplify the purification process, there are actually a number of other benefits

• A tag can add the ability to use a generic detection method (such as a standard tag-specific western-blotting antibody, or an enzymatic assay in the case of the GST-tag)
• It may actually help overcome some challenges with your protein e.g. by stabilizing it or making it more soluble, as shown in this (very cool :) example of how a challenging spider silk protein was purified by using a special solubility tag in addition to a Histidine-tag
• Some tags can be fused to proteins for a broad range of other applications such as—labeling for imaging and localization studies, protein–protein interaction studies, and subcellular localization or transduction
• Tags also allow strong binding to chromatographic affinity media in the presence of denaturants which makes it possible to purify a protein that requires this, e.g. if it has been expressed in inclusion bodies. In this case, you can then also try to perform refolding of the protein while it is still bound to the column (too complex to describe here, we will discuss this in-depth in a future post..)

Pros	Cons
Using a tag
Simple, generic purification using affinity chromatography (AC) as a first step	Tag may interfere with protein structure and affect folding and biological activity
Tags are easy to detect, in comparison to the target protein, which allows for a generic detection method	If tag needs to be removed, cleavage may not always be achieved at 100 % and sometimes some amino acids may be left
Solubility and stability can be improved	Only some tags can be used under denaturing conditions
Purifying a native protein
Tag removal is not necessary	The purification and detection protocols need to be designed specifically
Purification can always be done under denaturing conditions	Problems with solubility and stability may be   difficult to overcome and may need special protocols to be developed

       

In summary, tag if you can – it simplifies your purification work massively, but don’t be afraid to express and purify your protein in its native form. 

Let us know your experiences working with tags, preferences and if you have any questions in the comments. Next time, we will look at the pros and cons of the most common tags used for protein purification.