In the process of expression and obtaining of fusion proteins , one of the most critical and most challenging stages is the purification of recombinant proteins . This step is a special challenge when working with uncharacterized proteins, or with proteins for which there is no good antibody.
The tags affinity usually generally small amino acid sequences whose fusion to the target protein by genetic engineering techniques allows, in theory, the purification of any protein by affinity chromatography, without having prior knowledge of the biochemical properties Of the same.
In this post we bring you a compilation of the most used affinity tags for the purification of recombinant proteins.
The choice of the fusion tag that we want to add to our target protein will depend mainly on the purification priorities, the size of the tag and the cost of the chromatography resins.
Although the purification of recombinant proteins by affinity chromatography against fusion tags is generally used as a single step, in cases where higher purity is required this technique can be used as a preliminary step within a more complex purification procedure, or simultaneously fusing two tags to the 3 ‘and 5’ ends of the target protein, and carrying out a double affinity purification against each of them.
Below we summarize the characteristics of the affinity tags most used in the purification of recombinant proteins:
1.- HIS TAG
- Name : Polyhistidine
- Size : 6 aa
- Features :
- It is the most commonly used tag.
- Due to its small size, it rarely interferes with protein function.
- Likewise, the elution by means of the imidazole gradient is not very aggressive, which allows preserving the immunogenicity of the proteins.
- The purification of recombinant histidine tag proteins can be performed by IMAC (Immobilized Metal Affinity Chromatography), generally with Ni2 + columns, or by primary anti-polyhistidine antibodies.
- In the case of the proteins expressed in E. coli, purification by means of the histidine tag can reach a purity of 80%. However, in other systems such as insect or mammalian cells, the purity achieved may be lower due to the high percentage of histidine residues that the proteins present in these systems.
2.- GST TAG
- Name : Glutathione S-transferase
- Size : 26 kDa
- Features :
- Another of the most popular tags and one of the largest.
- It has the ability to increase the expression and solubility levels of the fusion protein.
- Due to its large size, it could interfere with the function of the protein.
- Glutathione elution is not very aggressive, which allows the functionality and antigenicity of the fusion protein to be preserved in most cases.
- The purification of recombinant GST tag proteins can be carried out using glutathione resins, and using anti-GST antibodies.
- The GST tag can be separated and removed from the fusion protein, while still attached to the resin.
- GST-bound proteins are generally expressed at high levels in E. coli, which can lead to the formation of aggregates in inclusion bodies.
3.- MBP TAG
- Name : Maltose-binding protein.
- Size : 45kDa
- Features :
- It is the largest tag.
- It has the ability to increase the levels of expression and solubility of the fusion protein, also contributing to its correct folding.
- Due to its large size, it could interfere with the function of the protein.
- Purification of recombinant MBP tag proteins can be purified using amylose resins and anti-MBP antibodies.
- The MBP tag cannot be separated and removed from the fusion protein while it is still bound to the resin, and the protein must be eluted prior to cleavage.
- As with the GST tag, MBP-bound proteins are generally expressed at high levels in E. coli, which can lead to the formation of aggregates in inclusion bodies.
4.- CBP TAG
- Name : Calmodulin-binding peptide
- Size : 26aa / 4kDa
- Features :
- Being a small tag, the probability that it affects the properties of the protein of interest is very low.
- One of the main advantages of this system lies in the mild binding and elution conditions, which contribute to the fusion protein maintaining its native form after purification.
5.- STREPTAVIDIN / BIOTIN BASED TAGS
- Name : BCCP (biotinylation signal peptide) + biotin
- Size : 100aa
- Features :
- Proteins that include the BCCP tag can be biotinylated by the enzyme biotin ligase.
- Once biotinylated, the purification of recombinant proteins can be carried out by affinity chromatography with avidin resin.
- While nonspecific binding to avidin in E. coli is very low, in other systems such as mammalian cells, proteins other than the target protein could be eluted simultaneously.
6.- EPITOPE TAGS
- Name : FLAG, Hemagglutinin (HA), c-myc, T7, etc.
- Size : 8 aa (FLAG), 31aa (HA), 11aa (c-myc), 11aa (T7)
- Features :
- Epitope tags are usually small sequences that act at the level of the epitopes of the protein of interest (antibody recognition sites).
- The purification of recombinant proteins with epitope tags can be performed by affinity chromatography, immobilizing specific primary antibodies.
- It is not the most recommended system in the case of highly insoluble proteins or those with a tendency to form aggregates.
In conclusion , the choice of the tag (and the cleavage method, if applicable) for the purification of recombinant proteins , should be made taking into account the particularities of our protein of interest. For proteins that are well expressed, the use of some of the simplest affinity tags such as polyhistidine tail would suffice, while in the case of those proteins that present greater expression difficulties, it would be necessary to resort to tags that also facilitate solubility and folding such as GST or MBP.
On the other hand, it should also be borne in mind that small tags hardly interfere with the structure, activity and characteristics of the target protein, while larger tags (GST, MBP) can have a significant impact both in the structure and in the biological activity of the fusion protein, and it would be necessary to assess in each case the desirability of cleaving said tag once the protein of interest has been purified.