GST Tags Overview

GST Tags for Protein Purification

GST affinity tag to aid in the purification of recombinant proteins. The 26KDa GST moiety binds with high affinity to glutathione coupled to a Sepharose matrix. This binding is reversible and the protein can be eluted under mild, non-denaturing conditions by the addition of reduced glutathione to the elution buffer. A specific protease site engineered between the GST moiety and the protein of interest allows removal of the GST moiety from the target recombinant protein. The GST can be removed from the sample by re-chromatography on a glutathione column, and the protein of interest purified to homogeneity by other techniques such as gel filtration or ion exchange.

Fig1. Flow diagram illustrating the key decision-making steps for designing and executing a successful GST fusion protein purification.
(Methods Mol Biol, 2011)

After protein expression and purification, it is necessary to determine whether to remove the tag according to different protein applications. If the tag is large, whether to remove it should be considered according to the downstream application. If the GST fusion portion is to be removed, it can be excised with a site-specific protease. The detection method can be detected by GST antibody or expressed target protein specific antibody.

GST fusion proteins can be detected by a colorimetric assay with the GST substrate 1-chloro-2,4-dinitrobenzene (CDNB) or with anti-GST antibodies. Precipitations of GST fusion proteins with glutathione-coupled beads are commonly used for the detection of protein-protein or protein-DNA interactions.