Researchers have a number of great tools at their fingertips for the purposes of protein purification and GST-tagging proteins is a popular choice. Glutathione S-transferase is a 26kDa protein encoded by the parasitic helminth Schistosoma japonicum and is commonly used by researchers as a protein tag in heterologous expression systems.
The addition of the GST tag to a protein of interest allows us to utilise GST’s high affinity for reduced glutathione to easily pull down the GST fusion protein from a cell sample. This is often done using a glutathione crosslinked agarose matrix to bind the GST fusion protein, requiring only a rinse with a reduced glutathione solution to yield a purified protein. Proteins tagged with GST are generally stably folded and expressed and the addition of a GST tag to certain proteins can often act as a molecular chaperone to stabilise their structure or even increase their expression.
Pull-down with GST tags has been used successfully to quantify protein levels within cells and assess protein-protein interactions, where appropriate antibodies or aptamers for use in pull-down to identify binding partners are often not available. Some interesting studies that have utilised this technique include the identification of the MAPK interactome, the validation of the use of transient SUMOylation to detect protein-protein interactions in vivo with proteins at near endogenous levels and the absolute quantification of transcription factors during the differentiation of adipocytes.
There are many commercial anti-GST antibodies available for use in downstream assays for the detection of the GST-tagged protein, such as ELISAs, Western blots and dot blots. However, despite their pronounced specificity the level of cross-reactivity exhibited by these antibodies is often high. Consequently some of these antibodies are extensively cross-adsorbed, to remove antibodies that bind to proteins other than the desired GST target. Yet while adsorption of an antibody may increase specificity, it can sometimes titrate out some of the antibody resulting in decreased sensitivity.
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