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GST-tagged Proteins – Production and Purification

The GST Tag

Glutathione S-transferase (GST) is a 211 amino acid protein (26kDa) 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 (26kDa) is fused to the N-terminus of the recombinant protein.

Because GST rapidly folds 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).

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Overview of Affinity Purification

Overview of Protein Expression

Overview of Pull-down Assays

GST Fusion Protein Purification

Glutathione is a tripeptide (Glu-Cys-Gly) that is the specific substrate for glutathione S-transferase (GST). When reduced glutathione (GSH) is immobilized through its sulfhydryl group to a solid support, such as crosslinked 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 the 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 26kDa, 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.

Related literature...

Protein Purification Handbook

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Pierce Glutathione Agarose Resins, Columns and Kits

Pierce Glutathione Superflow Agarose Resin

B-PER Bacterial Protein Extraction Reagents

GST Protein for controls

All fusion protein purification resins

HRV 3C Protease

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 microplates or anti-GST antibody plates enable fusion proteins to be captured from crude or semi-purified samples for plate and reporter assays of various kinds.
  • 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.

View products...

Glutathione Coated Microplates
Anti-GST Coated Plates

GST-Tag Protein Interaction
Pull-Down Kit

Active GTPase Pull-Down and Detection Kits

Search for Anti-Tag Antibodies
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Written and/or reviewed by Douglas Hayworth, Ph.D.

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