Purification and characterization of a recombinant human Theta-class glutathione transferase (GSTT2-2).
A cDNA encoding the human Theta-class glutathione transferase GSTT2-2 was expressed in Escherichia coli as a ubiquitin fusion protein. The co-translational removal of the ubiquitin by a cloned ubiquitin-specific protease, Ubp1, generates enzymically active GSTT2-2 without any additional N-terminal residues. The recombinant isoenzyme was purified to apparent homogeneity by DEAE anion-exchange, gel filtration, dye ligand chromatography and high resolution anion-exchange chromatography on Mono Q FPLC. The recombinant enzyme had significant activity with a range of substrates, including cumene hydroperoxide and 1-menapthyl sulphate. The activity of GSTT2-2 with a range of secondary lipid peroxidation products such as the trans,trans-alka-2,4-dienals and trans-alk-2-enals, as well as its glutathione peroxidase activity with organic hydroperoxides, suggest that it may play a significant role in protection against the products of lipid peroxidation.
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Division of Molecular Medicine, John Curtin School of Medical Research, Australian National University, Canberra, Australia.
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D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity; D014452:Ubiquitins / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; C491107:GSTT2 protein, human; D005982:Glutathione Transferase