Applications of fusion tags

Techniques used for structural studies, such as NMR spectroscopy, X-ray crystallography, and cryo-electron microscopy, require high-quality, pure protein for analysis₁. This means that purification methods that rely on detergents or reducing agents may not be suitable, as these can prevent the formation of good-quality crystals or affect protein structure₁.

Attaching a fusion tag to your protein of interest can enhance your ability to isolate your target by increasing yield, aiding solubility, and providing a known epitope to target. The downside for structural studies is that fusion tags can prevent the formation of well-ordered crystals and can be too large for NMR studies.

Cleavage of the tag from the protein can be a solution, but this increases experimental cost. Nevertheless, over 75% of proteins purified for crystallization are attached to fusion tags. Some tags are more suitable for structural studies than others, so it is important to consider your experimental design carefully.

Generation of a functionally active protein after purification can be critical for certain studies, especially those investigating potential therapeutics₁.  A major aspect of functional studies is the retention of correct signaling domains and accurate protein folding. Therefore, small tags may be preferred as these are less likely to affect protein function. The location of the tag is also critical for these studies, as incorrect placement can reduce correct expression. If the structure of your protein is known, this can aid optimum placement.

Both epitope and florescent tags are vital tools in assessing the localization of your protein of interest. Immunofluorescence microscopy is the most common technique for visualization, which can be achieved either directly through a fluorescent tag or indirectly through immunodetection. This can additionally be used to discover which regions of a protein contribute to localization₂.

Cells can also be fractionated before analysis and then analyzed for the fusion protein to help determine localization to specific subcellular compartments₂.

As fluorescent tags are non-toxic, they can be used in visualizing the movement of proteins in live cells.

If you wish to introduce a gene into a cell, fusion tags can be used to ensure that it is expressed. Co-expression of two genes can also be assessed, using either the same of different tags₂.

Recombinant proteins are also used to express eukaryotic proteins in prokaryotic systems. This technique is often used to achieve high levels of a protein of interest for subsequent study. One of the challenges associated with isolating eukaryotic proteins in this way is that 20–40% of them cannot be expressed in a soluble form in prokaryotic systems1. Fusion tags, such as GST or MBP, can help overcome this obstacle by increasing solubility of the protein of interest.

Protein purification relies on four basic steps: 

1. Lysis of the cell

2. Binding the protein of interest to a matrix

3. Washing off the unwanted lysate

4. Elution of your protein

Affinity chromatography, immunoprecipitation (IP), and protein complex immunoprecipitation (co-IP) are the most common techniques used for protein purification₂. Although these methods all utilize the four steps listed above, each technique uses a different stationary matrix.

Affinity chromatography commonly uses a column as the stationary phase and exploits certain molecular properties, such as hydrogen or ionic bonding. The crude lysate is first flowed through the column to allow binding of the protein of interest before the unwanted lysate is subsequently washed away. Your protein of interest can be eluted from the column by the addition of specific buffers or solutions.

On the other hand, the stationary phase of IP is an antibody against the protein of interest. The cell lysate is incubated with the stationary phase, allowing the antibody to bind to the protein in solution. The antibody/antigen complex can then be pulled out of the sample using protein A/G-coupled agarose beads for subsequent analysis, for example, separation by SDS-PAGE for western blot analysis.

Co-IP also uses antibodies to bind a known target, but the aim is to pull down your protein of interest along with any additional proteins that it is bound to. In this way, novel binding partners or protein networks can be found₁. To gain a complete understanding of the network of proteins associated with your primary known protein, follow up experiments often include the repetition of the co-IP using the proteins discovered in the additional pull-down as the target.

Although it can take some time to find the optimal tag for your protein of interest, we know it is good to have a starting point. Therefore, we have outlined some widely used tags to get you started, along with a comparison of their molecular weights [Figure 1].

Protein purification: His-tags, Biotin, c-myc

Protein expression: DDDK, CBP

Protein localization and detection: GFP, mCherry, HA

Immunoprecipitation: c-myc, DDDK

Protein localization: GFP, mCherry

Functional analysis: DDDK

Increasing protein solubility: GST