Three commonly used affinity tags for protein purification

In order to purify heterologous proteins from various hosts, affinity tags are extremely capable tools. In the recent years affinity tags have become highly popular tools for protein purification mainly due to following reasons. They provide high level of purification of recombinant proteins from crude extracts, mostly in a single step. Second, they provide mild elution conditions, thereby, do not interfere with the structure and hence the function of the purified proteins. In addition, affinity tags allow a variety of proteins to be purified using easy procedures.

Affinity tags are available as expression vector systems having multiple cloning sites (MCS) for cloning the gene of interest towards the N or C-terminal of the tag. Now-a-days, a variety of affinity tags are available for the purification of recombinant proteins. Each tag has its certain advantages and disadvantages. Here, you will see the properties of the three commonly used affinity tags: His-Tag, GST-Tag and MBP-Tag.

1)  His-Tag

His-tag is the most commonly used affinity tag for the purification of recombinant proteins in E.coli. The His-tag is a peptide motif that consists of six histidine (His) residues. Therefore, it is also called as hexa histidine-tag or 6xHis-tag. The most commonly used bacterial expression vector system for His-tag is the pET series (Novagen). These vectors provide both the N & C-terminal fusion of gene of interest with the His-tag.

It is observed that hexahistidine has high affinity towards transition metals e.g. Ni⁺⁺, Co⁺⁺ etc. Therefore, in order to purify recombinant proteins with hexa histidine-tag, immobilized-metal affinity chromatography (IMAC) columns are used. Ni-NTA agarose is the most commonly used resin for the purification of His-tag proteins. Since very few naturally occurring proteins bind to the Ni-NTA matrices with considerable affinities, therefore, recombinant proteins containing the His-Tag are significantly purified in a single step.

The recombinant protein is generally eluted either with the lowering of pH or with the addition of imidiazole to the column.

Advantages:

• Hexahistidine tag is much smaller and hence provides high yields of tagged proteins.
• It does not interfere with the structure and function of the recombinant protein.
• Its affinity for Ni-NTA matrix is non-dependant on the conformation of the target protein.
• The elution conditions for His-tagged protein are milder.
• Ionic strength, chaotropic agents and detergents do not affect the purification of His-tag proteins.
• Less expensive.

Because of all these advantages, His-Tag is the affinity tag of choice for various protein expression and purification experiments.

The only disadvantage of the tag is that certain bacterial proteins are able to bind to the His-Tag and hence are co-eluted with the recombinant protein. This can be partially ruled out by increasing the stringency of washing.

2)  GST-Tag

The Glutathione S-transferases (GSTs) are class of enzymes that are involved in cellular defense against small toxic compounds. They are abundant enzymes that utilize glutathione as a substrate. GSTs bind to glutathione with high affinity and specificity. Therefore, they provide an efficient system for the purification of proteins.

Generally, the Glutathione S-transferase (GST) from Schistosoma japonicum is used as the affinity tag in the pGEX vector series. The gene encodes a protein of 218 residues having molecular weight of 26KDa. The fusion protein thus produced can be purified using the glutathione-based affinity resins. The strength and selectivity of the resin for GST-tagged proteins results in the successful purification of the recombinant protein from the cell extract, in a single step.

In order to elute the recombinant proteins, reduced glutathione is added to the column. This allows the elution of recombinant proteins under mild and non-denaturing conditions.

Advantages:

• Provides a higher degree of purification in a single chromatographic step.
• Increases the solubility of the recombinant protein.
• It does not interfere with the structure and function of the recombinant protein.
• Provides immunogenic as well as biochemical assay of the recombinant protein.
• The elution conditions for GST-tagged protein is milder than most of the affinity purification methods.

Disadvantages:

• Due to its larger size it is prone to degradation by proteases.
• Affinity for glutathione resin depends on certain reagents.
• More expensive.
• In order to study the protein of interest in detail the tag has to be removed.

Nonetheless, GST-tag is a versatile tag for protein expression and purification. It is generally used when protein of interest is not expressed in His-tag system or is having solubility or purity issues.

3)  MBP-Tag

Maltose Binding Protein (MBP) is a periplasmic protein of the bacterium E.coli. The protein is a component of the maltose and maltodextrins. It is encoded by the gene malE. The malE gene product bears a wide variety of fusions and hence is suitable for expressing proteins which do have problems in expression, in His-Tag or GST-Tag systems.

The expression vector system consisting of MBP as the affinity tag is pMAL series (New England Biolabs).

As MBP is a component of maltose, therefore, the fusion protein can be purified using matrices consisting of sugars. Generally amylose resins are used for the purification of MBP tagged proteins. For the elution of the recombinant protein maltose is added to the column. As a result of this, the elution of recombinant proteins is mild and under non-denaturing conditions.

Advantages:

• Provides a higher degree of purification in a single chromatographic step.
• Increases the expression as well as solubility of the recombinant protein.
• It does not interfere with the structure and function of the recombinant protein.
• Allows periplasmic expression of the recombinant protein.
• Allows the formation of disulfide bonds in the foreign proteins.
• Limits degradation of the recombinant protein.
• Less expensive.

Disadvantages:

• Due to its larger size, expression of large proteins is sometimes problematic .
• In order to study the protein of interest in detail the MBP tag has to be removed

The elution conditions for MBP-tagged protein is milder than most of the affinity purification methods. In addition, due to bacterial origin of malE gene, MBP is a better tag in terms of expression and solubility as compared to GST.

These were the overview of the three most commonly used  affinity tags for the purification of heterologous proteins in E.coli. According to our experiences, one should start with the His-Tag, followed by GST and MBP. In most of the cases, His-Tag results in decent level of expression and purification of recombinant proteins. For any query or suggestions related to protein purification in E.coli, please feel free to post it in the comments.