The GST Tag
Glutathione S-transferase (GST) is a 211 amino acid protein whose DNA sequence is frequently integrated into expression vectors for production of recombinant proteins. The result is a GST-tagged fusion protein in which the functional GST protein (26 kDa) is fused to the N-terminus of the recombinant protein.
GST-tagged fusion proteins can be purified or detected based on the enzyme-tag's ability to bind its substrate glutathione. Alternatively, anti-GST antibodies are commercially available for use in assay methods involving GST-tagged proteins. In either case, the tag provides a means of specifically purifying or detecting the recombinant protein without a protein-specific antibody or probe.
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, 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.
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 often appear as a ladder of lower MW bands below the full-sized fusion protein.
Protein Purification Handbook
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
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.
Glutathione Coated Microplates
Anti-GST Coated Plates
GST-Tag Protein Interaction
Active GTPase Pull-Down and Detection Kits
Search for Anti-Tag Antibodies
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