Uses
Glutathione S-Transferase from human placenta has been used in antigen microarray and informatics analysis.
General Description
Glutathione S-transferase (GST) is a major detoxification enzyme, and exists as multiple cytoplasmic and membrane-bound isozymes. These isozymes differ in their catalytic activity, as well as in their non-catalytic binding properties. Cytoplasmic isoforms of GST are encoded by five genes, namely α, θ, μ, σ and π. α, μ and π are the most abundant forms in mammals. Membrane bound GST forms are encoded by a single gene.
Biochem/physiol Actions
Glutathione S-transferase (GST) catalyzes conjugation reactions during phase II metabolism. This enzyme also confers protection against catabolic products of peroxidized lipid and oxidized DNA, and ROS (reactive oxygen species) or electrophiles.
Purification Methods
It is purified by affinity chromatography using a column prepared by coupling glutathione to epoxy-saturated Sepharose. After washing contaminating proteins, the pure transferase is eluted with buffer containing reduced glutathione. The solution is then concentrated by ultrafiltration, dialysed against phosphate buffer at pH ~7 and stored in the presence of dithiothreitol (2mM) in aliquots at <-20o. [Simons & Vander Jag Anal Biochem 52 334 1977.]
Description
Recombinant Glutathione S-Transferase full length protein (1-218a.a.) expressed in E.coli, having a molecular mass of 26kDa. GST was isolated from an E. coli strain that carries the coding sequence for Schistosoma japonicum GST under the control of a T7 promoter.
The GST is purified by proprietary chromatographic techniques.
Background
Antioxidant enzyme Glutathione S- Transferase (GST) is thought to do the primary cellular defense mechanism against reactive oxygen species. GST reduces lipid hydroperoxides through its Se-independent glutathione peroxidase activity. The enzyme also detoxifies lipid peroxidation end products such as 4-hydroxynonenal (4-HNE).
The soluble GST is a 26 kDa protein which occurs as a dimer in all aerobic organisms. Each monomer has two domains, one that binds GSH and is an /-structure similar to thioredoxin and the other, all helical, that binds the hydrophobic substrate. The GST -fusion protein expression system is a widely used recombinant protein expression system that allows a peptide or a regulatory protein domain to be expressed as a fusion to the C-terminus of Schistosoma japonicum GST. Fusion proteins also possess GST -enzymatic activity and can undergo dimerization similar to in vivo. The fusion protein can be purified via GST -affinity column chromatography. In most cases, the desired peptides or domains are removed from GST by applying a specific protease that recognizes and cleaves the linker between the protein domain and GST. The technique has been widely used to generate different kinds of proteins for crystallization, molecular immunology studies, the production of vaccines and studies involving protein-protein and protein-DNA interactions.
