Question

What is glutathione-S-transferase (GST) and how it can be used as a protein “tag” for purification. Explain the association between principles of affinity chromatography and GST fusion and GST purification .

Answer 1

Glutathione is a tripeptide (Glu-Cys-Gly) which is a specific substrate for glutathioneS-transferase (GST). When reduced glutathione is inactive within its sulfhydryl group a strong support, such as cross-linked agarose, can be used to hold GST or GST-tagged proteins by enzyme-substrate reaction.
Binding is most effective in near neutral conditions (physiological conditions) such as Tris-buffered saline (TBS) at pH 7.5. Because binding depends on the preservation of the essential structure and enzymatic activity of GST, protein denaturants are not compatible.
After washing the conjugation column to remove bound sections of the sample, the purified GST-fusion protein can be dissected and recovered (eluted) in the glutathione column with the addition of reduced glutathione. Free glutathione eliminates the non-invasive interaction of glutathione with GST, which allows fusion proteins to occur in the affinity column.
This assimilation system usually yields over 90% of the GST-tagged recombinant pure from bacterial lysate samples. Purification used for Glutathione-based protein packaging based on GSTs was easily performed at a small, medium or large facility to produce the amount of microgram, milligram or gram.
At 26 kDa, GST is much larger than other protein tags. For reasons not completely mentioned in the literature, the formation of the GST-fusion tag is often contaminated in the expression change and reduction of protein gel electrophoresis (e.g., SDS-PAGE). Because of this, recombinant GST-fusion protein electrical samples tend to appear as a ladder for low MW bands under full mass protein.
When a GST tag is unwanted or unwanted as part of an interacting protein after purification, it may be removed if the cleavage site of a particular proteinase is included between the protein and the GST tag in the DNA vitamin construct.