interest will be expressed together as a fusion protein. Because the GST protein has a strong binding affinity for GSH, beads coated with the compound can be added to the protein mixture; as a result, the protein of interest attached to the GST will stick to the beads, isolating the protein from the rest of those in solution. The beads are recovered and washed with free GSH to detach the protein of interest from the beads, resulting in a purified protein. This technique can be used to elucidate direct protein–protein interactions. A drawback of this assay is that the protein of interest is attached to GST, altering its native state. A GST-tag is often used to separate and purify proteins that contain the GST-fusion protein. The tag is 220 amino acids (roughly 26 kDa) in size, which, compared to tags such as the Myc-tag or the FLAG-tag, is quite large. It can be fused to either the N-terminus or C-terminus of a protein. In addition to functioning as a purification tag, GST acts as a chaperone for the attached protein, promoting its correct folding, as well as preventing it from becoming aggregated in inclusion bodies when expressed in bacteria. The GST tag can easily be removed following purification by addition of thrombin protease if a suitable cleavage site has been inserted between the GST-tag and the protein of interest (which is usually included in many commercially available sources of GST-tagged plasmids). See also Affinity chromatography Bacterial glutathione transferase Glutathione S-transferase Mu 1 Glutathione S-transferase, C-terminal domain GSTP1 Maltose-binding protein Protein tag References Low et al 2007 External links Overview of Glutathione S-Transferases Preparation of GST Fusion Proteins GST Gene Fusion System Handbook Transferases