I. What Is a Protein Tag?

Protein tagging technology refers to the use of genetic cloning techniques to fuse peptides or protein domains with specific functions—or to fuse entire proteins with the target protein—in order to purify, quantify, and track the expression levels of the target protein.

II. How Do Affinity Tags Affect Fusion Proteins?

1. Increased expression levels of fusion proteins: In studies examining the binding and expression of affinity tags with target proteins, certain affinity tags located at the N-terminus of the target protein—such as Small ubiquitin-related modifier (SUMO) and glutathione S-transferase (GST)—typically result in a significant increase in the yield of recombinant proteins. Affinity tags provide a reliable translation start site, enabling ribosomes to efficiently translate methionine (at the N-terminus), thereby promoting the expression and production of fusion proteins.

2. Enhancing the solubility of fusion proteins: Currently, to overcome the drawback of inclusion body formation during recombinant protein expression in E. coli, one of the most extensively studied approaches is the fusion of an affinity tag with the target protein. Among these, the solubility-enhancing properties of the MBP tag have been studied in depth. By altering the “open” and “closed” conformational equilibrium of MBP, its solubility-enhancing capacity can be significantly influenced. This indicates that MBP’s solubility-enhancing properties are related to the “open” conformation of the protein itself.

3. Promotes proper protein folding: Whether a protein folds correctly depends primarily on its own amino acid sequence, while the affinity tag plays a role in facilitating proper folding. The optimal tag for different target proteins still requires verification through numerous experiments. Affinity tags can also control the degradation of bound proteins, thereby increasing the sensitivity of fusion protein binding assays.

III. Commonly Used Affinity Tags

1. Short Peptide Tags

1.1 Histidine Tag (His-Tag): Consists of 5–15 amino acid residues, with a molecular weight of approximately 0.84 kDa. Its purification ligands are immobilized metal ions: nickel, copper, zinc, and cobalt. Characteristics of the tag include: (1) a small number of tag molecules; (2) purification is possible in environments generated by non-ionic surfactants or under denaturing conditions; (3) Suitable for studying protein-protein and protein-DNA interactions; (4) Relatively low immunogenicity, allowing purified genes to be directly injected into animals for antibody production; (5) Compatible with various expression systems and suitable for mild purification conditions; (6) Can be used to construct dual-affinity tags.

1.2 FLAG Tag: Composed of eight amino acid residues (DYKDDDDK), with an anti-FLAG monoclonal antibody serving as the purification ligand. This tag possesses inherent hydrophilic properties. Due to its small molecular size, it neither masks the antigenic epitopes and domains of the target gene nor interferes with the function of the bound gene, and it can be cleaved by enterokinase. The three specific monoclonal antibodies for this tag are: M1, M2, and M5. M1 monoclonal antibody recognizes the N-terminal FLAG tag (in the presence of Ca²⁺); M5 monoclonal antibody binds to Met-FLAG-tagged fusion proteins but cannot recognize the C-terminal FLAG tag. M2 monoclonal antibody, on the other hand, can recognize and bind to the N-terminal Met-FLAG tag, the FLAG tag, and the C-terminal FLAG tag in the absence of Ca²⁺.

1.3 Strep-tag II: This tag can be fused to any position on a protein. Its purification conditions are relatively broad; fusion proteins can be eluted with 2.5 mmol/L desulfurized biotin and are not dependent on metal ions, making it suitable for proteins containing metal ions.

1.4 CBP tag: Composed of 26 amino acid residues and weighing approximately 4 kDa, it uses solid-phase calmodulin as the ligand for purification. The tag’s characteristics are: (1) it can be fused to either the N-terminus or C-terminus; (2) it can be removed by thrombin or enterokinase; (3) in low-concentration calcium buffer, it can be selectively captured and bound by calmodulin resin, while in a neutral environment, it can be eluted with 2 mM EGTA, making the reaction relatively mild.

1.5 c-Myc Tagging Method: Consisting of eleven amino acid residues, this method has been successfully applied in Western blot hybridization, immunoprecipitation, and flow cytometric quantification techniques, and can be used to monitor the expression of recombinant proteins in target cells.

2. Protein Tags

2.1 GST Tag (Glutathione S-Transferase): It consists of 211 amino acid residues and has a molecular weight of approximately 26 kDa; glutathione resin is used as the purification ligand. The GST tag not only improves the solubility of fusion proteins to a certain extent but also increases protein yield, facilitating subsequent purification. Furthermore, the GST tag offers mild purification conditions, efficient translation, and low-cost affinity media, making it one of the most commonly used tags today.

2.2 MBP Tag: Composed primarily of 396 amino acids and with a molecular weight of only 40 kDa, it plays a crucial role in the soluble expression of recombinant genes in prokaryotic systems. Experimental results show that maintaining a balance between the “open” and “closed” conformations of MBP significantly alters the solubility of the MBP tag. This means that once the MBP tag assumes the “open” conformation, it can specifically bind to affinity resin during the purification process. The MBP tag can be fused to either the N- or C-terminus of a protein and can be specifically recognized and removed by enterokinase.

1.5 Halo Tag: Consists of 300 amino acid residues, with a molecular weight of 33 kDa; the purification ligand is an alkane chloride. Labeling characteristics: (1) Can be fused to the N- or C-terminus of a gene; (2) Enables the detection and affinity binding of the target gene to a solid-phase immobilized resin; (3) The target ligand must be a small-molecule compound capable of covalent fusion with the Halo-Tag in vitro or in vivo.

3. Other tags

IV. Combined Use of Affinity Tags 

1. His6-MBP: The bound protein is the His6-MBP target protein. The His tag is primarily used for affinity purification of the bound protein, while the MBP tag is mainly used to enhance the solubility of the protein during expression. A specific recognition cleavage site for the tobacco etch virus protease is inserted into the middle of the target gene for the combined tag, which facilitates tag removal.

2. Tandem Affinity Purification: Without disrupting the target protein’s regulatory sequences, the TAP tag (protein tag) is inserted at one end of the target protein. Through a two-step affinity chromatography process, specific protein complexes that are close to their natural state are obtained, and the proteins are identified using mass spectrometry.

V. Applications of Fusion Tags

1. Protein Purification: The most common use of tags. For example, the His-Tag is widely used for the purification of E. coli proteins and endogenous cellular proteins.

2. Western Blot Analysis: Protein expression can be monitored using small-molecule tags with matching antibodies. The FLAG-Tag has a low molecular weight and is available with many commercially available antibodies, making it an important tag for Western blot experiments.

3. Immunoprecipitation: Commonly used tags include the FLAG tag, HA, and cMyc.

4. Live-Cell Imaging: Fluorescent proteins (FPs) are commonly used as marker proteins in live-cell imaging.

    KMD Bioscience has been dedicated to research on recombinant protein expression and purification for many years. We have designed a set of expression vectors containing various fusion tags (such as His, GST, FLAG, and SUMO) to ensure high-solubility protein expression and high protein activity, thereby guaranteeing the quality of every recombinant protein we produce and providing our customers with superior service.