GST Tag - How a protein helps to purify a protein

 
GST tagged protein
Fig. 1: A GST Tagged protein
 


What is the GST tag?

There are multiple protein affinity tags that are being used for protein purification and the GST Tag is one of them. GST is short for Glutathione S-transferase and referes to a whole protein, not just a few amino acids like the Rho1D4 tag. The GST tag has been in use for protein purification since the late 1980s (Smith & Johnson 1988) and has been established as a reliable way for protein purification assays that required highest level of purity (Harper & Speicher 2013).

The size of GST Tag is 26 kDa and therefore really large compared to other protein affinity tags. In theory it can be fused to either the C- or N-Terminus of a protein (Terpe 2003), but we highly recommend to add it to the N-Terminus. So far it has been successfully used to purify proteins from bacterial cells (Smith and Johnson 1988), yeast (Lu et al. 1997) and mammalian cells (Rudert et al. 1996). Its general features & sequence can be found here.

So we know now, that the GST Tag basically is a whole protein that gets attached to the protein of interest. But what is the Glutathione-S-Transferase now exactly?


What is GST / Glutathione-S-Transferase?

Glutathione S-Transferases are a familiy of procaryotic and eukaryotic enzymes that are mainly involved in the detoxification of the cells and body. In the most extreme cases GST proteins can make up to 10 % of the whole cytosolic proteome of some mammalian cells (Oakley 2011). The reaction Glutathion S-Transferases are catalyzing is the oxidation of reduced Glutathione, also called GSH. GST's main function in the cell is the protection of DNA and cellular proteins from external toxic substances. The mechanism does by catalyzing a nucleophilic attack onto the toxic substance. Through the activation of a thiol group of GSH, that then reacts with suitable groups on the toxic substances. This coupling of GSH to the toxins leads to their excretion from the cell or further metabolic procession (Hayes et al. 2004). The high specificity of GST towards reduced Glutathione (GSH) is the reason for the high protein puritiy of the purification assays.
GST detoxification of the cell Fig. 2: Process of detoxification by Glutathatione S-Transferases
As stated: There are numerous GST orthologs out there, but a specific one was chosen to be the template for the GST Tag. So why GST from Schistosoma japonicum?

Why GST from Schistosoma japonicum?

Glutathione-S-Transferases can be found in organisms ranging from procaryots to eucaryots (Allocati et al. 2008). So why is this specific GST protein used for affinity purification? What are its advantages? This answer derives back to the first description of GST as an option for affinity chromatography and protein purification. As formally cited, the first mention of GST for protein purification was in 1988 by Smith & Johnson. Before that Smith et al. published a paper in which they descripted the possible use of GST as a vaccine against Schistosoma japonicum.
Schitosoma sp. Fig. 3: 3-D Rendering of Schitosoma
Schistosoma japonicum, a platyhelminthes is a major cause for the disease schistosomiasis and the GST protein of said species showed promise to be a the base for a vaccine against Schistosoma japonicum (Smith et al. 1988). Because of this study Smith already worked with this specific ortholog of the GST protein, and therefore it remained the base for the later described protein purification method. And since 1988 GST from Schistosoma japonicum, remained as the GST ortholog that is used as the GST affinity tag.


GST Tag features

We compiled the most important data regarding the GST tag and its capabilities for protein purification.
Note: We listed the "standard" sequence used in pGEX vectors.

Table 1: Overview over the most important features of the GST tag
Features of the GST Tag:
Amino Acid Sequence MSPILGYWKIKGLVQPTRLLLEYLEEKYEE
HLYERDEGDKWRNKKFELGLEFPNLPYYID
GDVKLTQSMAIIRYIADKHNMLGGCPKERA
EISMLEGAVLDIRYGVSRIAYSKDFETLKV
DFLSKLPEMLKMFEDRLCHKTYLNGDHVTH
PDFMLYDALDVVLYMDPMCLDAFPKLVCFK
KRIEAIPQIDKYLKSSKYIAWPLQGWQATF
GGGDHPPKSD
DNA Sequence
(Based on pGEX-4T1 vector)
ATG TCCCCTATAC TAGGTTATTG GAAAATTAAG
GGCCTTGTGC AACCCACTCG ACTTCTTTTG
GAATATCTTG AAGAAAAATA TGAAGAGCAT
TTGTATGAGC GCGATGAAGG TGATAAATGG
CGAAACAAAA AGTTTGAATT GGGTTTGGAG
TTTCCCAATC TTCCTTATTA TATTGATGGT
GATGTTAAAT TAACACAGTC TATGGCCATC
ATACGTTATA TAGCTGACAA GCACAACATG
TTGGGTGGTT GTCCAAAAGA GCGTGCAGAG
ATTTCAATGC TTGAAGGAGC GGTTTTGGAT
ATTAGATACG GTGTTTCGAG AATTGCATAT
AGTAAAGACT TTGAAACTCT CAAAGTTGAT
TTTCTTAGCA AGCTACCTGA AATGCTGAAA
ATGTTCGAAG ATCGTTTATG TCATAAAACA
TATTTAAATG GTGATCATGT AACCCATCCT
GACTTCATGT TGTATGACGC TCTTGATGTT
GTTTTATACA TGGACCCAAT GTGCCTGGAT
GCGTTCCCAA AATTAGTTTG TTTTAAAAAA
CGTATTGAAG CTATCCCACA AATTGATAAG
TACTTGAAAT CCAGCAAGTA TATAGCATGG
CCTTTGCAGG GCTGGCAAGC CACGTTTGGT
GGTGGCGACC ATCCTCCAAA ATCGGATCTG
GTTCCGCGTG GATCCCCGGA ATTCCCGGGT
CGACTCGAGC GGCCGCATCG TGACTGA
Size 26 kDa
Compatibility to recombinant proteins N-Terminus is highly recommended
Combinable with other affinity tags? Yes, see our GST-and-Rho-Tagged eGFP as an example
Specificity of interaction (KD) 1 µM
Typical protein yield per ml of high quality purification resin. 10-12 mg/ml
Elution conditions Reduced Glutathione , pH shift or tag cleavage
Binds to purification resin / magnetic bead via Reduced Glutathione
Suitable Protein Purification Methods Agarose Resin & Magnetic Beads

How to add / clone a GST Tag into a plasmid

The best way to add a GST tag to a protein of interest is using a vector of the pGEX series. They also come with different protein cleavage sites and included protein cleavage sites. These are important for the later mentioned methods to remove a GST tag. Figure 4 depicts the process and steps from our recommended cloning strategy.

1. The pGEX vectors all come with a multiple cloning site (MSC) that contains multiple restriction enzyme digestion sites. These vary from pGEX vector to vector. Table 2 provides an overview here. In addition, all pGEX vectors contain three stop codons that cover all three possible reading frame on a DNA strain, after the MSC. This is to ensure that the expressed protein does not have to contain the stop codon by itself.

2. The next step is to create a PCR product of your gene / protein of interest that is flanked in both directions with restriction sites that are present in the MCS of the chosen pGEX vector. These primers should cover each:
  1. 3-4 nts overhang (the longer, the better for restriction digest, but we suggest 3-4)
  2. The restriction site
  3. 20-25 nts of your gene of interest
GST Tag cloning strategy Fig. 4: Overview of the cloning procedure for adding a GST Tag to a protein of interest.
Let us give you an example for this: Lets try to assume that you want to add a GST Tag to eGFP.

Legend - Forward primer:
Overhang
Restriction site -> BamHI in this case
First 20 nts of eGFP

5'-GGG GGATCC ATGAAACATCACCATCACCA-3'
Legend - Reverse Primer:
Overhang
Restriction site -> EcoRI in this case, reverse complement
Last 20 nts of eGFP, reverse complement
5'-GGG GGATTC TTTGTATAGTTCATCCATGC-3'
Please note that this presented strategy is only the one example of how a GST tag can be added to a protein of interest. Other methods like homologous recombination in yeast or bacteria are also possible ways to accomplish this.
Table 2: Different pGEX vectors and the restriction sites inside their MCS's, sorted in 5' to 3' direction.
pGEX vectorRestriction sites in 5' to 3' direction
pGEX-1λT BamHI , EcoRI
pGEX-2T BamHI, SmaI, EcoRI
pGEX-2TK BamHI, SmaI, EcoRI
pGEX-4T-1 BamHI, EcoRI, SmaI, SalI, XhoI, NotI
pGEX-4T-2 BamHI, EcoRI, SmaI, SalI, XhoI, NotI
pGEX-4T-3 BamHI, EcoRI, SmaI, SalI, XhoI, NotI
pGEX-3X BamHI, SmaI, EcoRI,
pGEX-5X-1 BamHI, EcoRI, SmaI, SalI, XhoI, NotI
pGEX-5X-2 BamHI, EcoRI, SmaI, SalI, XhoI, NotI
pGEX-5X-3 BamHI, EcoRI, SmaI, SalI, XhoI, NotI
pGEX-6P-1 BamHI, EcoRI, SmaI SalI, XhoI, NotI
pGEX-6P-2 BamHI, EcoRI, SmaI SalI, XhoI, NotI
pGEX-6P-3 BamHI, EcoRI, SmaI SalI, XhoI, NotI


How does GST Tag protein purification work?

GST Tag protein purification is an affinity chromatography method. It exploits the high affinity of GST towards reduced Glutathione, its natural substrate. Fo achieve this Glutathione is bound to either agarose resin or magnetic beads, depending on the prefered purification method. Figure 5 shows a schematic depiction of such a bead.

The actual purification protocol differs from supplier to supplier of GST Tag affinity products. These differences can also include different buffer compositions that are only optimized for their own specific GST affinity beads. Therefore we highly recommend not to apply protocols for GST Tag protein purification from one supplier to another supplier's product.

You can find Cube Biotech's GST Tag affinity products here.

The corresponding protocols are listed here:
Agarose Resin GST Tag purification protocol:

Magnetic Bead GST Tag purification protocol:
GST Affinity Beads schematic Fig. 5: Schematic depiction of a GST Tag affinity bead. Through a flexible spacer on the beads surface the Glutathione tripeptide is bound to the beads and can be used to bind GST tagged proteins.


GST Tag protein purification protocol

The protein purification with GST affinity beads is presented in figure 5. These core steps are based on the purification procedure using GST Tag affinity magnetic beads.

Step 1: The cell lysate after cell lysis. The whole proteome of the cell floats inside the lysis buffer. GST Tagged proteins are unseparated from the other proteins.

Step 2: Addition of GST Tag Affinity MagBeads to the cell lysate. More MagBeads should be added than GST Tagged protein is suspected inside the cell lysate.

Step 3: Rotation of the cube / flask for a couple of hours to give the glutathione beads enough time to bind all GST Tagged proteins inside the cell lysate.

Step 4: GST Tagged proteins have now bound to the MagBeads. Due to the specificity of the GST tag to the Glutathione on the beads no other proteins should have bound.

Step 5: Application of a magnetic force onto the cell lysate. The GST tagged proteins have now bound to the Glutathion beads, while no other protein is has.

Step 6: Removal of the unbound cell lysate / protein. The GST Tagged proteins are now purified.
GST Tag protein purification workflowFig. 6: Overview of the workflow of an exemplary GST Tag protein purification using magnetic beads.
To verify the success of a GST Tag purification, we recommend to perform a Western Blot that uses Anti-GST antibodies to detect the purified protein. An Western Blot example protocol can be found here.


How to remove a GST Tag?

With 26 kilodalton (kDa) the GST Tag is one of the largest affinity tags out there. As it is infact a whole protein, it can even surpass some proteins that it gets attached to. So of course this creates the valid concern that an artificial attachment of this size to a protein can interfere with said protein's functionality. Therefore the question arrises if it is possible to remove a GST Tag after protein purification to ensure that it does not hinder the purified protein in any way.

This concern was already in the mind of the creators of the pGEX vector series, that we already mentioned in table 2. By creating a GST Tag fusion protein in the way as described above in the chapter "How to add / clone a GST Tag into a plasmid" you get the option to remove this GST Tag later on.

The answer is to add a specific protein cleavage site in 5' direction of the MCS of the plasmid. This way the N-Terminal GST Tag can be cleaved off, even after purification. This can also be used as an elution method during purification instead of the elution buffer that is mentioned in our agarose resin and magnetic bead purification protocols.

Since this method includes a small chance that your protein of interest includes a protease cleavage site by coincidence there are three different options of protein cleavage sites that are already present in the pGEX vectors.
Table 3: Different pGEX vectors and their included protease cleavage site.
pGEX vectorProtease Cleavage site in 5' of the MCS
pGEX-1λT; pGEX-2T; pGEX-2TK; pGEX-4T-1; pGEX-4T-2; pGEX-4T-3 Thrombin
pGEX-3X; pGEX-5X-1; pGEX-5X-2; pGEX-5X-3 Factor Xa
pGEX-6P-1; pGEX-6P-2; pGEX-6P-3 PreScission Protease


GST Tag Removal protocol (example)

Note: This protocol describes how to cleave off a GST Tag using the thrombin cleavage site from free eluted GST fusion proteins.

Table 4: Required reagents for Thrombin cleavage
ReagentAmount
Thrombin Depends on supplier: 3-10 U of thrombin can cleave 1 mg of fusion protein at RT overnight on average. Still read the supplier's notes
1 X PBS buffer 140mM NaCl; 2.7mM KCl; 10mM Na2HPO4; 1.8mM KH2PO4; pH 7.3
PMSF Enough for a 1 mM concentration in your protein solution
AEBSF Alternative to PMFS; Enough for a 1mM concentration in your protein solution


Procedure (general version)

  1. Add to the eluate or column purification 10 cleavage Units of thrombin ~10 µl per milligramm of GST fusion protein
  2. Mix gently and incubate the mixture of room temperature (22°C) for 2-16 hours.
  3. After the digest you can stop the reaction by either adding 1 mM of PMSF or ABESF
  4. Check the Results via SDS page OR
  5. Directly separate the cleavage products by SDS chromotography (e.g. Size exclusion chromatography)
If you have found your statisfying cleavage conditions you can scale up the procedure in a linear way.

Tip: You can incubate a sufficient amount of your GST tagged protein to see on SDS-Page gels with 0.01 to 0.06 units of thrombin and different digestion times (e.g. 2h, 4h, 6h etc.). Stop the reaction as descripted abd jeep the alinqots at -20°C until use.

References

1.
Hayes, J. D., Flanagan, J. U. & Jowsey, I. R. GLUTATHIONE TRANSFERASES. Annu. Rev. Pharmacol. Toxicol. 45, 51–88 (2004).
2.
Allocati, N., Federici, L., Masulli, M. & Di Ilio, C. Glutathione transferases in bacteria. The FEBS Journal 276, 58–75 (2009).
3.
Oakley, A. Glutathione transferases: A structural perspective. Drug metabolism reviews 43, 138–51 (2011).
4.
Smith, D. B. et al. Mr 26,000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione S-transferase. Proc Natl Acad Sci U S A 83, 8703–8707 (1986).
5.
Terpe, K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology 60, 523–533 (2003).
6.
Rudert, F. et al. pLEF, a novel vector for expression of glutathione S-transferase fusion proteins in mammalian cells. Gene 169, 281–282 (1996).
7.
Harper, S. & Speicher, D. W. Purification of proteins fused to glutathione S-transferase. Methods Mol Biol 681, 259–280 (2011).
8.
Smith, D. B. & Johnson, K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31–40 (1988).
9.
Lu, Q., Bauer, J. C. & Greener, A. Using Schizosaccharomyces pombe as a host for expression and purification of eukaryotic proteins. Gene 200, 135–144 (1997).
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