GST Tagged Protein and GST Tag Purification

What is GST Tag?

GST Tag Definition

Glutathione-S-transferase (GST), a 26 kDa sequence of 211 amino acids, is another widely used affinity tag that increases solubility of the desired protein. GST tag has affinity for immobilized glutathione and is used for prokaryotic expression more frequently. It can be fused to either the N-terminus or C-terminus of a protein.

Glutathione Affinity is an efficient method for single-step purification of proteins fused to a GST (glutathione S-transferase) tag. GST can be expressed as a soluble protein in the E. coli cytoplasm in high amounts and with full enzymatic activity. Furthermore, many eukaryotic proteins that are insoluble when expressed in E. coli have been shown to be at least partially soluble when expressed as a GST fusion protein.

GST Tagged Protein Purification

How to Purify GST Fusion Proteins

To generate constructs that express GST fusion proteins, the sequences coding for the protein of interest can be inserted into commercially available vectors. In addition to its use for affinity purification, the GST tag is frequently utilized in so-called pull-down experiments to investigate protein–protein interactions.

The scale of purification of GST-tagged proteins is dependent on the amount of protein in the preparation. A column size and total binding capacity should be chosen to approximately match the amount of protein to be purified. Very few non-tagged proteins are usually retained on the resin if the target protein occupies nearly all of the available glutathione-binding sites (GST fusion sites). If too much matrix is used, other proteins may bind nonspecifically to unoccupied sites.

In GST fusion protein purification, GST tagged proteins contained in a cleared lysate bind to immobilized glutathione (GSH, Bind). And nonbinding proteins are washed from the matrix (Wash) and bound GST fusion proteins eluted from the support by the addition of excess reduced glutathione (Elute).

GST Fusion Protein Purification with Affinity Chromatography

Affinity chromatography is one of the most selective types of chromatography, and it can be a very useful technique for protein purification. A convenient method of protein expression and subsequent purification is to fuse a protein with a glutathione-S-transferase (GST) domain. The general purification strategy is thus to bind the GST fusion protein on a column of immobilized glutathione, wash away all the other stuff, and then elute the protein.

There are four main steps to purify a GST fusion protein:
1. Solutions to prepare Buffer A for glutathione column
2. Glutathione column purification
3. Evaluation of purification
4. Removal of glutathione on a Nap-10 column

Besides affinity purification, other applications for GST-tagged fusion proteins are made possible with the aid of glutathione-ligand chemistries or GST-tag-specific antibodies such as Microplate coating, Protein interaction pull-down and ELISA or Western blot.

GST Tagged Protein Purification FAQs

Why use a GST-tag for protein expression and production?

Adding a GST-tag to a protein's N-terminus can facilitate its detection, isolation, and purification by GSH affinity resin. More importantly, since GST is highly expressed protein with excellent solubility, fusing a difficult to express protein to the GST tag sometimes can greatly enhance the recombinant protein's expression as well as it solubility.

How do I cleave of the GST-tag after purification?

In some applications, it is desirable to remove the GST-tag, for example, for protein crystallization. To allow cleavage of the GST-tag, a protease cleavage site needs to be engineered between the tag and the protein. An EK cleavage site behind the GST-tag (GST-EK site-protein structure) can allow complete removal of the GST-tag and the cleavage site, leaving no additional amino acids after the specific cleavage of the GST-tag. For more information on the cleavage site and tag removal by EK and HRV-3C protease, please refer to: Enterokinase (EK), HRV-3C (human rhinovirus protease).

References

Kimple ME, et al. (2013) Overview of affinity tags for protein purification. Curr Protoc Protein Sci 73: 9.9.1-9.9.23.
Xu ML, et al. (2017) Two-step chromatographic purification of glutathione s-transferase-tagged human papillomavirus type 16 e6 protein and its application for serology. Protein Expr Purif 132: 19-26.

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