Protein Tags: A Comprehensive Overview

1. What Is A Protein Tag?

A protein tag is a peptide or protein that is genetically fused to either the N- or C-terminal of the protein of interest during the cloning step for a specific purpose. These tags vary in size, ranging from just a few amino acid residues to full-length proteins or domains.

2. Functions of Protein Tags

As a type of important tool in biological research, protein tags confer the target protein with additional properties such as affinity, solubility, and fluorescence, and thus widely used in multiple fields, including protein purification, protein detection and quantification, solubility enhancement, protein yield improvement, subcellular protein localization, protein tracing, and protein-protein interaction research ^[1].

Protein tags are useful in recombinant protein production. Different protein tags may execute distinct functions but may be overlapping:

Enhance protein affinity and solubility ^[2,8];

Increase the expression of target proteins ^{[9, 13]};

Facilitate the isolation and purification of target proteins;

Protect target proteins from degradation ^[10-12];

Augment the folding ability of target protein ^[14].

Protein tags can be recognized by specific antibodies, affinity resins, or other binding partners, enabling researchers to manipulate and study the tagged protein with greater ease and precision.

3. Common Protein Tags

There are a variety of available protein tags in the market. According to their properties, protein tags can be roughly divided into affinity tags, epitope tags, and fluorescent tags. The following table lists commonly used protein tags, protein tag comparisons, as well as protein tag advantages and disadvantages.

Table 1. Commonly used protein tags

4. How to Select An Appropriate Protein Tag

Selecting an appropriate protein tag depends on the specific requirements of the conducted experiment or application. Here are some factors to consider when choosing which protein tag to use in your research system:

Purpose of the fusion: Determine whether the purpose of adding protein tags is, for example, detection, purification, localization, or other. Generally, His-tag and Flag-tag are preferred for protein purification and Western blot, respectively. Flag, HA, and cMyc tags can be selected for immunoprecipitation, and GFP and CFP protein tags are used for immunofluorescence purposes.

Protein tag size: Whether the target protein needs a larger or smaller tag depending on its application. In theory, the larger the added protein tag, the greater the impact on the function and structure of the target protein. Small tags are useful for protein detection and antibody generation since they are less immunogenic than larger tags ^[5]. Common tags with low molecular weight include 6*His, Flag, HA, and c-Myc, which have only a few amino acids and rarely affect the structure and function of the protein.

Required production levels: Higher levels of protein production are needed in structural studies, which can be quickly attained by using a larger fusion tag with robust translational initiation signals, while the investigation of physiological interactions calls for lower production levels and smaller tags ^[1].

Tag location: Protein tags placed at the N-terminus or C-terminus of the target protein can promote different effects. Typically, N-terminal tagging is more beneficial than C-terminal tagging. N-terminal tags are friendly for efficient translation initiation, enabling fusion proteins to exploit the tag's efficient translation initiation sites. Most endoprotease cleavage at or near the C-terminus of their recognition sites leaves none or a few additional residues at the native N-terminal sequence of the target protein after N-terminal tags are removed ^[1,6].

However, If you do not have the exact protein structure or protein functional domain map, it is recommended to construct both N-terminal tagged and C-terminal tagged expression clones to test which one is more effective.

Protein tag removal: Every protein tag, whether large or small, may interfere with downstream functions of the target proteins. So, protein tag cleavage from the target proteins is necessary whenever it is feasible. To accomplish this, specific protease cleavage sites are placed in between the tag and the target protein. Site-specific proteases function in the cleavage site, releasing the target protein in its natural form. Two kinds of proteases endoproteases and exoproteases can be used to detach tags.

Table 2. Common endoproteases for tag removal ^[7]

5. How to Tag A Protein

Once an appropriate protein tag has been selected, primers with sequences complementary to the upstream region of the gene of interest and the desired protein tag sequence are designed.

Amplify the target gene using PCR with the designed primers, incorporating the protein tag sequence at the 5'- or 3' -end based on the experiment goal.

Clone the PCR product into a suitable expression vector using restriction enzyme digestion and ligation or Gibson assembly.

After cloning, the resulting vector contains the target gene fused with the protein tag gene at either the N- or C-terminal. The tagged vector is transfected into a suitable host organism for protein expression. The presence of the specific tag enables efficient and thorough purification of the tagged protein, giving rise to highly pure proteins.

Due to their potential to impact the structure or activity of the target protein, protein tags are often removed from their fusion partners using highly sequence-specific proteases for downstream biological and functional studies.

6. Protein Tag-associated Products

CUSABIO provides many tag antibodies that are helpful for the recognition and detection of tagged proteins.

Conclusion

Protein tags genetically fused to a target protein have revolutionized detection and purification procedures, enabling researchers to study protein function, localization, and interactions with high precision. By understanding the diverse range of protein tags available, researchers can tailor their experiments to achieve optimal results.

References

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