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.