When performing protein expression, the selection of appropriate tags must be made according to the experimental objectives and protein characteristics. The choice of tag directly impacts the efficiency of both protein expression and purification, as the use of tags can significantly streamline the protein purification process. Selecting a well-suited tag is crucial for ensuring smooth progress in experiments.

1、What are the commonly used tags in protein expression?

Commonly used protein tags include 6xHis, GST, SUMO, Flag, and Strep-II Tag.

2、What are the characteristics of these tags?

(1) 6xHis: Immobilized metal affinity chromatography (IMAC) can be used for the separation and purification of recombinant proteins.

-- Its inherent characteristics do not affect the target protein and it does not form dimers.

-- With a small molecular weight of only 0.84 kDa, it does not interfere with downstream applications of the protein.

-- It has low immunogenicity, allowing purified proteins to be directly injected into animals for immunization and antibody production.

-- It is compatible with various protein expression systems, and its mild purification conditions have minimal impact on the protein.

(2) GST (Glutathione S-transferase): GST is a transferase that plays an important role in detoxification processes.

-- Enhances protein solubility and increases expression yield.

-- Preserves the antigenicity and bioactivity of the protein, improving the stability of foreign proteins.

-- Can be expressed in various host systems, offering broad applicability.

-- High specificity, enabling convenient and mild purification.

-- The large molecular size (~26 kDa) may interfere with protein function and downstream experiments.

(3) SUMO：A small ubiquitin-related modifier protein, it is a highly conserved class of large proteins involved in protein sumoylation and present in eukaryotes.

--As a fusion tag for recombinant protein expression, it possesses molecular chaperone functionality.

--It promotes the correct folding of proteins.

--It exhibits tolerance to heat and proteases, further aiding in maintaining the stability of the target protein.

--The SUMO tag has a dedicated protease (highly specific) that recognizes the tertiary structure of SUMO, enabling extremely precise cleavage without leaving any residual amino acids, making it suitable for recombinant protein expression.

(4) Flag：The artificially designed fusion tag features a hydrophilic sequence composed of six amino acids (Lys-Asp-Asp-Asp-Asp-Lys) near the C-terminus, which may form a highly exposed three-dimensional protein conformation. Theoretically, this allows the sequence to achieve maximal hydrophilicity, thereby significantly enhancing the antigenicity of the Flag fusion tag.

-- Does not affect the function and properties of the target protein.

-- The sequence is short and can be encoded by a single synthetic oligonucleotide strand.

-- During crystallization, the conformation of the Flag fusion protein is almost identical to that of the target protein alone. It typically does not interact with the target protein and generally does not affect its properties or function, facilitating downstream research on the fusion protein by researchers.

-- The corresponding Flag tag antibody can be used for the detection and purification of the fusion protein.

-- Significantly improves the purification efficiency of the target protein.

(5) Strep-II Tag：The binding affinity between Strep-tag and Strep-Tactin is at least 100 times stronger than that of natural streptavidin.

-- Does not mask other protein epitopes or domains in the fusion protein.

-- Less likely to alter the function, secretion, or transport of the fusion protein.

-- The purification conditions are flexible, and the fusion protein can be rapidly eluted using 2.5 mM desthiobiotin.

-- The performance of the Strep-Tag II system remains unaffected even in the presence of chelating agents, detergents, reducing agents, or salts up to 1 M in the solution.

3、How to choose a protein tag?

If the protein is highly hydrophilic and prone to forming inclusion bodies, the SUMO tag or GST tag is recommended to promote soluble expression.

(1) The SUMO tag can be considered here because it comes with a specific protease (highly selective) that recognizes the tertiary structure of SUMO. This protease exhibits extremely high cleavage specificity and leaves no residual amino acids, making it suitable for recombinant protein expression.

(2) The GST tag is relatively large, and whether to remove it should be determined based on downstream applications. If the GST fusion portion needs to be removed, site-specific proteases can be used for cleavage. Detection can be performed using either a GST antibody or an antibody specific to the expressed target protein.