There are a number of great methods in the researcher’s toolbox for protein purification. Along with the ever popular His-Tagged protein purification, there is also the choice of GST-tagged proteins. Glutathione S-transferase is a super family of enzymes that have been utilized by researchers to create the GST gene fusion system. Due to GST’s high affinity for reduced glutathione, it is incredibly easy to pull the GST fusion protein (combined with your protein of interest) from a cell sample. For that purpose, many researchers use a glutathione crosslinked agarose resin as a matrix for binding to the GST fusion proteins. A simple rinse with a reduced glutathione solution and a pure protein sample is yours!
Of course, there are always some limitations. The GST tag is comparatively large, around 26 kDa (the His-tag is around 1 kDa), though it is not as large as Protein A (30 kDa) or the Maltose binding protein (40 kDa). But many commercially available GST tags usually contain a convenient cleavage domain which can then be used to remove the purification tag with a great deal of specificity (just make certain you don’t have a similar site on your protein of interest!). It is also almost always fused to the N-terminus of the protein, which may limit your ability to purify it depending on your protein’s structural configuration. But generally, proteins are stably folded when fused with the GST tag. It has further advantages in stabilizing small or unfolded protein sequences! And there are also many commercially available anti-GST antibodies for use in downstream assays involving your GST tagged protein.
GST is also very susceptible to denaturing, leading GST-tagged proteins to display a ladder of lower MW bands after an SDS-Page protein gel. In fact, purification of a GST fusion is all but impossible in the presence of even very low concentrations of SDS. But what if your protein has to be solubilized in SDS? Elodie Boisselier et al. developed a very interesting strategy to do just that. They found that the addition of 2-methyl-2, 4-pentanediol (MPD) to the protein mixture before running it through a column containing SDS proved sufficient to blocking the adverse effect of the SDS on the GST protein!
Regardless of your specific protein research needs, our goal at Gold Bio is to continue to support you with the quality reagents (and tips!) you might need to get the job done. And if you have any questions, please contact us at [email protected].
Douglas, Kenneth T. "Mechanism of action of glutathione-dependent enzymes." Adv Enzymol Relat Areas Mol Biol 59 (1987): 103-167.
Oakley, Aaron. "Glutathione transferases: a structural perspective." Drug metabolism reviews 43.2 (2011): 138-151.
Production of HexaHistidine or Glutathione-S-transferase (GST) tagged proteins in Escherichia coli (Ponnambalam Lab, Leeds)
Elodie Boisselier, et al. “A strategy for purifying glutathione S-transferase in the presence of sodium dodecyl sulfate.” BioTechniques 51.3 (2011): 193–194
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