Tagging your protein of interest can be extremely useful since it simplifies the purification protocol, improves the yield and solubility of your protein of interest and promotes the proper folding of their fusion partners. Due to their versatility, affinity tags (peptide sequences that are appended to the target protein) are recognized as one of the most powerful tools that can be used for basic biological research and in structural and functional proteomics as well.
As you may very well know by now, you need to obtain enough concentration of your protein of interest before you can study its function, structure and interactions with other proteins. While a lot of methods have been used to enrich proteins of interest from crude biological extracts, researchers agree that affinity purification (the process of purifying proteins by virtue of its specific binding properties to an immobilized ligand) is still the most effective method for this purpose.
In addition to purifying recombinant proteins, affinity tags are now also being used in western blot, immunoprecipitation (IP), immunohistochemistry (IHC), flow cytometry (FCM), and a number of other applications.
Affinity Tags in Protein Purification: When Should You Use Them?
Do you need to use a tag every time you purify your proteins? Well, this may depend on your purpose. Basically, you may use a tag if you don't need your protein in its native form.
However, if your downstream application warrants the use of a native protein, you may use a tag in the purification process and then cleave it later using a sequence specific protease, or not use a tag at all to preserve its native condition. Generally speaking, affinity tags are not used in cases where their presence will negatively affect the intended application of the protein (i.e. for clinical use).
Note: Small-size affinity tags exert minimal effect on the structure, activity, and characteristics of the recombinant protein so you can choose not to remove them. The 6× His, FLAG, Strep II, and CBP tags belong to this group.
Large-size tags such as maltose-binding protein (MBP) and glutathione s-transferase (GST) can have a positive effect on protein solubility and expression efficiency. However, they can have a significant impact on the structure and biological activity of their partner protein so you may need to cleave them using the appropriate protease.
Using Tags in Purifying Your Protein of Interest: The Pros and Cons
As mentioned earlier, using affinity tags greatly simplifies the protein purification process. In addition, here are some other benefits that you can enjoy by using them.
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Easy detection. Compared to your target protein, protein tags are significantly easier to detect.
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Improved protein stability and solubility. Using a protein tag such as MBP and/or GST can stabilize your protein of interest and improve its solubility.
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Binding to chromatographic affinity media. By using tags, you can purify proteins even under denaturing conditions and/or refold them while they are still bound to the chromatography column.
On the other hand, here are some of the most apparent drawbacks of using tags in purifying your protein of interest.
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It may affect the structure and function of your protein of interest. There is a great chance that the tag will affect the folding and biological activity of the protein.
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It may be difficult to cleave. There are times when you cannot achieve complete cleavage of the tag from your protein of interest. It is also possible that some amino acids may be left in the process.
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Not all tags can be used under denaturing conditions. You should know which tags can be used under denaturing conditions to ensure accurate results.
