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Recombinant Protein Expression: How to Choose the Right Affinity Tag

Posted in: Protein Expression and Analysis
An assortment of paper gift tags to represent protein purification affinity tags

So, you have selected a protein for biochemical studies and have cloned it into a tag-containing vector. After successfully expressing your protein in a soluble form, all you need to do is pick it out from the proteome pool of the expression system, such as E. coli or insect host cells.

An affinity tag, just like an identity card, allows you to identify and separate your protein from hundreds of others (a VIP status, sort of!). Protein purification affinity tags enable your protein to bond with resin-attached molecules, leaving behind other, non-tagged proteins (no tag, nothing to brag).

This short introduction to commonly used affinity tags will help you select the best one for your experimental conditions and obtain a joyful, “proteinaceous” reward for your hard work.

6X-His (hexahistidine) Tag

Hexahistidine tags are one of the most commonly used affinity tags. It utilizes the ability of histidine to coordinate with transition metal ions, such as Ni2+, Co2+, and Cu2+. Owing to its small size, it is non-toxic and does not interfere with the immunogenicity or physiochemical properties of the protein.

One should proceed with caution if using denaturing agents, such as urea, or reducing agents, such as dithiothreitol (DTT), as they may disturb the metal-ion resin.

A 6x-His tag is a great option for experiments involving a prokaryotic expression system. I achieved >95% purity of a cytoplasmic protein from Leishmania sp. in a single step using a Ni-nitrilotriacetic acid (NTA) resin. Moreover, you can regenerate and re-use the resin numerous times (a boon during fund crunch in the lab), given that conditions are amenable during each purification cycle.

Glutathione-S-transferase (GST) Tag

This tag works on the principle of GST’s affinity for immobilized glutathione. A GST tag can increase the solubility of your expressed protein in a prokaryotic system. You can remove the 26-kDa tag while the fusion protein is still bound to the glutathione resin, or you can elute the tagged protein. Tag removal is recommended as it may affect the properties of the fusion protein.

The elution procedure is mild, thus preserving the protein’s immunogenicity and properties. GST-based purification gives good yields. However, if your purification procedure requires the use of reducing agents, such as DTT or \beta-mercaptoethanol (\beta-ME), denaturing agents, such as urea or guanidine HCl, or detergents, GST-based chromatographic purification may be less effective.

Watch out for co-elution of an unwelcomed guest (70-kDa E. coli chaperonin) with your protein. In such a situation, treat the lysate with 5 mM MgCl2 and ATP before eluting.

Maltose-Binding Protein (MBP)

The main aim of this tag is to increase the solubility of the fusion protein, thus it may be your tag of choice when purifying membrane or lipophilic proteins. MBP-based purification helped me obtain decent expression and yield of a membrane protein that was difficult to achieve with a 6X-His-tag.

This tag gives a good yield; however, the size of the MBP tag (46 kDa) could be a concern if your downstream application is immunization. In such a scenario, you should consider removing the tag to avoid altering the protein’s immunogenicity.

The MBP-fusion protein bound to an amylose resin is eluted by running maltose in the elution buffer. This method is fairly resistant to denaturants, reducing agents, and detergents. However, any amylase activity in the cell lysate may reduce the efficiency of the amylose resin.

Calmodulin-Binding Protein (CBP)

This procedure utilizes the C-terminal domain of myosin light-chain kinase (MLCK) as a purification tag. In the presence of low levels of calcium, CBP has a high affinity for resin-attached calmodulin. Combined with superaffinity, smaller (4 kDa) size, and mild elution conditions, CBP tags are good if you are dealing with a delicate or finicky protein.

It can withstand low levels of notorious (yet sometimes essential) components such as denaturants, detergents, and reducing agents.

Streptavidin/Biotin-Based Tag

Who is not aware of the great affinity that biotin and streptavidin have for each other? Well, you can utilize it for protein purification by biotinylating your protein of interest and making it ‘pretty’ in avidin’s eyes (avidin-attached resins)!

Non-specific binding during purification is negligible in this method; however, the use of denaturing agents is a big ‘no-no’. This is widely used for immobilizing biotinylated fusion proteins on surface plasmon resonance (SPR) chips for functional studies.

Strep-Tag® Peptide System

This is a modification of the streptavidin/biotin-based purification system. The target protein is fused to a short peptide (9 amino acids) known as Strep-tag, which binds to a streptavidin. The protein is eluted by using biotin in the elution buffer. This system achieves a high purification efficiency even with low-expressing proteins.

The Strep-Tactin®/Strep-tag II system achieves even higher purification efficiency by utilizing engineered forms of streptavidin and an eight-peptide tag. This system is free from the drawbacks associated with metal-ion based resins, which can cause protein aggregation and protein precipitation by chelation. Additionally, you can reuse the resin multiple times.

Once you are finished with your purification, you can always choose to remove the tag, depending upon downstream experiments/assays. So, go ahead and choose a tag based on your protein’s comfort and liking.

Feel free to share your experiences working with the protein purification affinity tag of your choice in the comments below. I wish you good luck in achieving high protein yields!


  1. Kimple M.E., et al. Overview of affinity tags for protein purification. Curr Protoc Protein Sci, Chapter 9, Unit 9.9 (2013). doi: 10.1002/0471140864.ps0909s36
  2. Zhao X., et al. Several affinity tags commonly used in chromatographic purification. J Anal Methods Chem, 581093 (2013). doi: 10.1155/2013/581093
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