GST-tagged Proteins – Production and Purification

Protein purification with affinity tags such as glutathione S-transferase (GST), histidine (HIS), and other affinity tags, enables purification of proteins with both known and unknown biochemical properties. Therefore, this methodology has become a widely used research tool for determining the biological function of uncharacterized proteins. GST is a 211 amino acid protein (26 kDa) whose DNA sequence is frequently integrated into expression vectors for production of recombinant proteins. The result of expression from this vector is a GST-tagged fusion protein in which the functional GST protein (26 kDa) is fused to the N-terminus of the recombinant protein.

Because GST folds rapidly into a stable and highly soluble protein upon translation, inclusion of the GST tag often promotes greater expression and solubility of recombinant proteins than expression without the tag. In addition, GST-tagged fusion proteins can be purified or detected based on the ability of GST (an enzyme) to bind its substrate, glutathione (GSH).

GST-fusion protein purification

Glutathione is a tripeptide (Glu-Cys-Gly) that is the specific substrate for glutathione S-transferase (GST). When reduced glutathione is immobilized through its sulfhydryl group to a solid support, such as cross-linked beaded agarose, it can be used to capture pure GST or GST-tagged proteins via the enzyme-substrate binding reaction.

Binding is most effective in near-neutral buffers (physiologic conditions) such as Tris-buffered saline (TBS) pH 7.5. Because binding depends on preserving the essential structure and enzymatic function of GST, protein denaturants are not compatible.

After washing an affinity column to remove non-bound sample components, the purified GST-fusion protein can be dissociated and recovered (eluted) from a glutathione column by addition of excess reduced glutathione. The free glutathione competitively displaces the immobilized glutathione binding interaction with GST, allowing the fusion protein to emerge from the affinity column.

This affinity system commonly yields greater than 90% pure GST-tagged recombinant protein from crude bacterial or mammalian cell lysate samples. Glutathione-based affinity purification of GST-tagged fusion proteins is easily done at either small, medium or large scales to produce microgram, milligram or gram quantities.

At 26 kDa, GST is considerably larger than many other fusion protein affinity tags. For reasons that have not been fully characterized in the literature, the structure of the GST-fusion tag often degrades upon denaturation and reduction for protein gel electrophoresis (e.g., SDS-PAGE). As a result, electrophoresed samples of GST-fusion proteins often appear as a ladder of lower MW bands below the full-sized fusion protein. 

When the GST tag is not required or desired as part of the recombinant protein after purification, it can be removed if a cleavage site for a specific protease is included between the protein and GST tag in the design of the DNA vector. For example, HRV 3C protease specifically cleaves the sequence Leu-Glu-Val-Leu-Phe-Gln-↓-Gly-Pro

Other GST-tagged protein techniques

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:

• Microplate coating:  Glutathione-coated or anti-GST antibody microplates may be used for determining the presence and concentration of GST or GST-fusion proteins from cell lysates. Because the plates are pre-blocked, initial purification of the cell lysates is not necessary. Also, using these plates, which are available in both 8-well and 96-well formats, to immobilize GST-fusion proteins is useful for screening sera for antibodies to the fusion protein.  

• Protein interaction pull-down: Specific GST-tagged proteins and glutathione agarose resin are the basis of kits designed to purify, identify and measure specific protein interaction complexes.
• ELISA or western blot detection: Anti-GST antibodies are available for these applications.

   

Frangioni, J.V. and Neel, B.G. (1993). Solubilization and purification of enzymatically active glutathione s-transferase (pGEX) fusion proteins. Anal Biochem 210:179-87. 

Simons, P.C. and VanderJagt, D.L. (1977). Purification of glutathione S-transferases for human liver by glutathione-affinity chromatography. Anal Biochem 82:334-41.