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.