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Protein expression purification tags

Update time : 2020-11-10

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Protein expression purification tags
Some peptides and proteins are widely used in the mass production of recombinant proteins. They are fused and expressed with the target protein to facilitate the expression, detection, tracing and purification of the target protein. Such peptides or proteins are called protein tags. Generalbiosystems can use a variety of protein expression tags according to customer needs, including His, Sumo, GST, MBP, etc., to provide customized protein expression and purification services.

6*His Tag:

6*His refers to a fusion tag composed of six histidine residues, which can be inserted at the C-terminus or N-terminus of the target protein. 

As a tag, one is to form an epitope to facilitate purification and detection; the other is to form a unique structural feature (binding ligand) to facilitate purification. 

The side chain of histidine residues has a strong attraction to solid nickel and can be used for immobilized metal chelation chromatography (IMAC) to separate and purify recombinant proteins.

The use of His-tag has the following characteristics:

1. The molecular weight of the tag is small, only ~0.84KD, which generally does not affect the function of the target protein;

2. His tag fusion protein can be purified in the presence of non-ionic surfactants or under denaturing conditions. The former is used in the purification of highly hydrophobic proteins, and the latter is particularly useful in the purification of inclusion body proteins. 

After the denaturant is dissolved, the impurity protein is removed by metal chelation affinity chromatography, so that the renaturation is not interfered by other proteins, or the metal chelation affinity chromatography is renaturated;

3. His tag fusion protein is also used in the study of protein-protein and protein-DNA interactions;

4. His tag has relatively low immunogenicity, and the purified protein can be directly injected into animals for immune preparation of antibodies;

5. It can be applied to a variety of expression systems, with mild purification conditions;

6. It can be used with other affinity tags to construct parental affinity tags.

GST Tag:

GST (glutathione sulfhydryl transferase) tag protein itself is a transferase that plays an important role in the detoxification process, and its natural size is 26KD. It is mainly used in prokaryotic expression. 

There are roughly two reasons for its application in prokaryotic expression. 

One is because it is a highly soluble protein, and it is hoped that it can be used to increase the solubility of foreign proteins; the other is that it can be used in the large intestine A large amount of expression in bacillus plays a role in increasing the amount of expression.

The GST fusion expression system is widely used in the expression of various fusion proteins, and can be expressed in host cells such as E. coli and yeast. 

The bound fusion protein was eluted with 10 mM reduced glutathione under non-denaturing conditions. In most cases, the fusion protein is soluble in aqueous solution and forms a dimer.

GST tags can be easily detected by enzymatic analysis or immunoassay. Tags help protect recombinant proteins from degradation by extracellular proteases and improve their stability. In most cases, the GST fusion protein is completely or partially soluble.

Purification: The GST-tagged protein expressed by the expression system can be directly purified from the bacterial lysate using an affinity resin containing reduced glutathione (Glutathionesepharose). 

GST-tagged proteins can be eluted under mild, non-denaturing conditions, so the antigenicity and biological activity of the protein are retained. 

GST will lose its ability to bind to glutathione resin under denaturing conditions, so strong denaturing agents such as guanidine hydrochloride or urea cannot be added to the purification buffer.

If you want to remove the GST fusion part, you can use site-specific protease excision.

Detection: GST antibody or expressed target protein specific antibody can be used for detection.


SUMO tag protein is a small molecule ubiquitin-like modifier (Smallubiquitin-likemodifier), and it is one of the important members of the ubiquitin polypeptide chain superfamily. 

In the primary structure, SUMO and ubiquitin have only 18% homology, but the tertiary structure and biological functions of the two are very similar. 

Studies have found that SUMO can be used as a fusion tag and molecular chaperone for recombinant protein expression. 

It can not only further increase the expression of fusion protein, but also has the functions of resisting protease hydrolysis, promoting the correct folding of the target protein, and improving the solubility of the recombinant protein.

In addition, SUMO has an important application, that is, it can be used to completely remove the tag protein to obtain natural protein. 

Because SUMO proteolytic enzyme can recognize the complete SUMO tag protein sequence, and can efficiently cut SUMO from the fusion protein. 

After removing the SUMO, after affinity chromatography, the tag protein part is removed, and the recombinant protein is the same as the natural protein. 

Therefore, the SUMO tag is also often used in conjunction with other tags, as a specific enzymatic digestion site.

MBP Tag:

The MBP (maltose binding protein) tag protein has a size of 40kDa and is encoded by the malE gene of E. coli K12. MBP can increase the solubility of fusion proteins overexpressed in bacteria, especially eukaryotic proteins. The MBP label can be easily detected by immunoassay. It is necessary to cleave the tag with a site-specific protease. If the protein is expressed in bacteria, MBP can be fused to the N-terminus or C-terminus of the protein.

Purification: The fusion protein can be further purified by cross-linked starch affinity layer. The bound fusion protein can be eluted with 10 mM maltose in physiological buffer. The binding affinity is in the micromolar range. Some fusion proteins cannot bind effectively in the presence of 0.2% Triton X-100 or 0.25% Tween 20, while other fusion proteins are not affected. The buffer condition is pH 7.0 to 8.5, and the salt concentration can be as high as 1M, but denaturants cannot be used. If you want to remove the MBP fusion part, you can use site-specific protease excision.

Detection: MBP antibody or expressed target protein specific antibody can be used for detection.