I. What is Protein Tag?

Protein Tag is a technology that uses gene cloning to purify, measure and trace the expression level of target proteins by cloning peptides or protein structural domains with specific functions or by fusing complete proteins with target proteins.

II. What is the Effect of Affinity Tags on Fusion Proteins?

1. Increase the expression of fusion proteins: In the binding expression study of affinity tags and target proteins, if some affinity tags are at the N-terminus of the target proteins, such as small ubiquitin-related modifier (SUMO), glutathione S-transferase (GST), and other affinity tags, the fusion proteins will be more effective in the expression of fusion proteins. transferase (GST), etc., are usually able to significantly increase the yield of recombinant proteins. Affinity tags provide a reliable translation initiation site that enables the ribosome to efficiently translate methionine (N-terminal), thus promoting the expression and production of fusion proteins.

2. Enhance the solubility of fusion proteins: Currently, to overcome the shortcomings of E. coli expression of recombinant proteins that are prone to produce inclusion bodies, the relatively more researched method is the fusion expression of affinity tags with target proteins. Among them, the solubility of MBP tags has been studied in depth. By changing the "open" and "closed" equilibrium of MBP conformation, the auxiliary solubility of MBP can be significantly affected. This suggests that MBP's pro-solubility is related to the "open" conformation of the protein itself.

3. Promote the normal folding of proteins: The normal folding of proteins mainly depends on their amino acid sequence, and affinity tags play the function of helping normal folding. The optimal labeling of different target proteins needs to be verified through numerous experiments. Affinity tags are also capable of controlling the degradation of bound proteins, thus increasing the sensitivity of fusion protein binding analysis.

III. Commonly Used Affinity Tags

1. Short Peptide Tags

1.1 Histidine tag: consists of 5-15 amino acid residues, about 0.84 kDa. Its purification ligands are solidified metal ions: nickel, copper, zinc, and cobalt.

Tag Properties:

(1) small number of tag molecules

(2) can be purified in an environment free of ionic surfactant generation or a denaturing environment

(3) protein-protein and protein-DNA interaction studies

(4) relatively low immunogenicity, which allows for direct injection of purified genes into animals for immunization and preparation of antibodies

(5) can be used in multi-expression systems, with mild purification environments

(6) constructs biparental and tags

1.2 FLAG tag: composed of eight amino acid residues (DYKDDDDK), the purified ligand is an anti-FLAG monoclonal antibody, the tag has a natural hydrophilic group properties, because of the small molecular size, so it can not mask the antigenic epitopes and structural domains of the target genes, but also does not interfere with the function of the binding genes, and it can be resected by intestinal kinase. The three specific monoclonal antibodies for this marker, are M1 monoclonal antibody, M2 monoclonal antibody, and M5 monoclonal antibody.M one monoclonal antibody can recognize the N-terminal FLAG tag (in the presence of Ca2+); whereas, M five monoclonal can bind Met-FLAG-tagged fusion proteins, but is unable to recognize the C-terminal FLAG tag. In contrast, M II monoclonal antibody was able to recognize and bind to the N-terminal Met-FALG tag, FLAG, and the C-terminal FLAG tag in the absence of Ca2+

1.3 Strep-tag II tag: the tag can be fused to any position of the protein, the purification conditions of the tag are broader, the fusion protein can be eluted with 2.5 mmol/L desulfurized biotin, and it does not depend on metal ions, so proteins containing metal ions are suitable for tagging.

1.4 CBP tag: it consists of 26 amino acid residues, about 4KDa, and the ligand used for purification is solid phase calmodulin. The labeling properties are: (1) binding to either the N-terminal or C-terminal; (2) can be eliminated by thrombin and enterokinase; (3) can be differentially captured and absorbed by calmodulin resin in low-strength calcium buffer, and at the same time, it can be eluted by 2 mM EGTA in a neutral environment, and the reaction is relatively mild to the environment.

1.5 c-Myc tag: consisting of eleven amino acid residues, it has been successfully used in WB hybridization, immunoprecipitation, and flow cytometry to monitor the expression of recombinant proteins in target cells.

2. Protein Tags

2.1 GST tag (glutathione sulfotransferase): it consists of 211 amino acid residues, the size of about 26KDa, and the ligand for purification is glutathione resin. the GST tag not only improves the solubility of fusion proteins to a certain extent but also increases the protein yield, which is conducive to subsequent purification. Meanwhile, the GST tag has become one of the commonly used tags at this stage because of its mild purification conditions, efficient translation, and low cost of affinity medium.

2.2 MBP tag: mainly composed of three hundred and ninety-six amino acid groups, the size is only 40KDa, which is important for the soluble expression of recombinant genes in prokaryotic systems. Experimental results show that maintaining a balance between the "open" and "closed" conformations of MBP greatly changes the solubility of MBP tags, which means that when MBP tags are in the "open" conformation, the solubility of MBP tags is greatly altered, which means that when MBP tags are in the "open" conformation, the solubility of MBP tags is greatly altered. This means that when the MBP tag is in the "open" conformation, it can be specifically bound to the process of the affinity resin material. the MBP tag can be fused to the N/C-terminus of the protein and can be specifically recognized and removed by enterokinase.

2.3 Halo tag: It consists of 300 amino acid residues, 33KDa, and the purification ligand is alkyl chloride.

Tag Characteristics:

(1) can be fused to the N-terminal or C-terminal of the gene;

(2) can be found and affinity-bound solid-phase immobilization of the target gene;

(3) the target ligand must be a small molecule compound that can be covalently fused to the Halo-Tag in vitro or in vivo.

3. Other Tags

IV. Combined Use of Affinity Tags

1. His6-MBP: The combined protein is His6-MBP target protein, and its His tag is mainly used for affinity purification of the combined protein, while the MBP tag is mainly used to improve the soluble expression of the protein. The specific recognition cleavage site of tobacco etch virus protease was added in the middle of the combined labeled target gene, which is more conducive to the removal of the label.

2. Tandem affinity purification: Without destroying the regulatory sequence of the target protein, the TAP tag (protein tag) is embedded in one end of the target protein, and the specific protein complex close to the natural state is obtained through two-step affinity chromatography, and the protein is identified by mass spectrometry.

V. Application of Fusion Tags

1. Protein purification: the most common use of tags. For example, His-Tag is widely used in the purification of E. coli proteins and intracellular proteins.

2. Western Blot: Protein expression can be monitored by small molecule markers with matching antibodies. FLAG-Tag has a low molecular weight and many commercially available antibodies to match it, so it has become an important marker used in WB experiments.

3. Immunoprecipitation reaction: commonly used tags are FLAG-Tag, HA and cMyc.

4. Live cell images: Fluorescent Proteins (FPs) are commonly used as labeling proteins in live cell images.

KMD Bioscience has been devoted to recombinant protein expression and preparation research for many years, and has designed a set of expression vectors containing a variety of fusion tags (tags such as His, GST, FLAG, SUMO, etc.) to ensure highly soluble protein expression and high protein activity.