Tandem affinity purification

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.

Cleavage of your tag from your 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.

Enterokinase

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.

Factor X

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)

Cleavage site: Recognizes the tertiary structure of SUMO. Cleaves at N-terminus of the fusion protein.

Advantages: Can be used in a wide range of buffer conditions (pH 5.5–10.5) and temperatures (4–37°C). Can be used in 2 M urea, which aids the purification of SUMO-tagged proteins in inclusion bodies.

Limitations: Efficacy depends on the sequence of your protein of interest, for example, efficacy is reduced if there is a proline after the cleavage site.

Tobacco etch virus (TEV) protease

Cleavage site: ENLYFQ ^ S

Advantages: Widely recombinantly produced ensuring specific cleavage at a relatively low cost. Can be used at low temperatures and in a wide range of buffers. The residue after the cut site is flexible so the native N-terminal residue of your protein of interest can be used without loss of efficacy⁶.

Limitations: Wild type TEV protease can cleave itself, reducing efficiency.

Thrombin

Cleavage site: LVPR ^ GS

Advantages: Relatively specific, can be used in the presence of detergents. Efficiently removed by benzamine sepharose.

Limitations: Cleavage can occur at non-specific sites, although rarely and usually due to contaminants in commercial preparations.

3C and PreScission™ (human rhinovirus)

Cleavage site: ETLFQ ^ GP

Advantages: Optimal activity at 4°C

Limitations: Reduced efficiency at higher temperatures.

Inteins are another approach. These are segments of proteins that autocatalytically excise themselves from their host proteins. While abolishing the need for proteases, this method has limitations. First, inteins involve large regions of catalytic machinery, which increases the metabolic burden on the cells and does not have any positive impact in terms of enhancing solubility or ease of purification. Second, it can be a slow process and has not been highly tested in a high-throughput context. Finally, the efficiency of cleavage is dependent on the sequence context at the fusion junction⁵.

An effective affinity tag combines a chitin-binding domain (CBD) with a modified intein sequence. Although the elution step requires proteolytic cleavage, the binding between the tag and resin is very strong⁷.

References

1. Kimple, M.,, Brill, A.,, Pasker, R. Overview of Affinity Tags for Protein Purification Current Protocols in Protein Science 73 , (2013)

2. Kosobokova, E.,, Skrypnik, K.,, Kosorukov, V. Overview of fusion tags for recombinant proteins Biochemistry (Moscow) 81 ,187-200 (2016)

3. Young, C.,, Britton, Z.,, Robinson, A. Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications Biotechnology Journal 7 ,620-634 (2012)

4. Terpe, K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems Applied Microbiology and Biotechnology 60 ,523-533 (2003)

5. Waugh, D. Making the most of affinity tags Trends in Biotechnology 23 ,316-320 (2005)

6. Kapust, R.,, Tözsér, J.,, Copeland, T.,, et al. The P1′ specificity of tobacco etch virus protease Biochemical and Biophysical Research Communications 294 ,949-955 (2002)

7. Mitchell, S.,, Lorsch, J. Protein Affinity Purification using Intein/Chitin Binding Protein Tags Laboratory Methods in Enzymology: Protein Part D ,111-125 (2015)