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Download your in-depth PDF guide here. From determining the best application to identifying common issues, our guide will help you make the most of your bench time.
Not all tags are the same: they have different strengths and limitations. There are occasions where you may need a tag that can make your protein more soluble but also require a tag for purification. This is achieved by a process known as tandem affinity purification (TAP). TAP originally referred to fusing a specific series of domains onto a protein: a calmodulin-binding peptide (CBP) and Protein A from Staphylococcus aureus (ProtA), separated by a tobacco etch virus (TEV) cleavage site1. However, the process can now be used to fuse a variety of 2–3 tags onto your protein of interest, although one of the original TAP components is commonly included. These tags can either be added in tandem to one end of your protein or fused to each terminal. If the tag needs to be cleaved after use, fusing the tags in tandem is usually recommended for ease of cleavage.
His-tags are a popular choice for TAP due to their small size and effectiveness in purification. They are often combined with the maltose-binding protein (MBP), which provides an increase in solubility and reduces the likelihood of aggregates forming in E. coli systems2. Another common combination is using GFP and His, as this allows the detection of your protein via fluorescence methods3.
Although there are many advantages to using TAP tags, they are large, which could disturb protein function. Additionally, if you are using a CBP-containing tag, this can interfere with calcium signaling1. As each tag confers a specific advantage, there may be a tendency to want to add multiple tags to your protein. A good rule of thumb is that you do not want more tag than protein of interest.
Some tags infer little risk to protein functionality but others, especially those of large size, can have downstream implications. In these cases, it is often desirable to cleave the tag from your protein of interest after the initial detection. This can be achieved by using site-specific proteases or inteins.
Site-specific proteases should not affect the functionality of your protein, but it is often desirable to remove the protease after cleavage. One solution is to use a protease that is itself fused to an affinity tag. The protease can then be easily removed via affinity chromatography4. Several protease recognition sites exist depending on your specific need, each with distinct advantages and limitations. One point to consider is whether the tag is fused to the N- or C-terminal.
Cleavage of a tag from the N-terminal will leave minimal excess residues on your protein of interest. On the other hand, cleavage of C-terminal tags will result in 4–6 extra residues being left on your protein of interest. However, in limited circumstances, carboxypeptidases can be used to remove these short C-terminal sequences1. We have outlined the most common protease sites below.
Cleavage site: DDDDK^X
Advantages: Internal recognition site present in the DDDK (FLAG®) tag
Limitations: Variable efficacy dependent on the amino acid after the lysine residue, ranging from 61% (X = proline) – 88% (X = alanine). Cleavage can occur at non-specific sites.
Cleavage site: I(E/D)GR^X
Advantages: Widely available.
Limitations: Amino acid “X” cannot be an arginine or proline. Factor X can bind calcium ions and, therefore, should not be used with chelating agents, such as EDTA. Cleavage can occur at non-specific sites.
SUMO protease (S. cerevisiae Ulp1)