Expression, purification, and biochemical characterization of SAG, a RING finger redox-sensitive protein

We recently reported the cloning and characterization of SAG (sensitive to apoptosis gene), a novel zinc RING finger protein, that is redox responsive and protects mammalian cells from apoptosis. Here we report the expression , purification, and biochemical characterization of SAG. Bacterially expres sed SAG is brown in color and dithiothreitol (DTT)-sensitive. SAG forms lar ge oligomers without DTT that can be reduced into a monomer in the presence of DTT. These features help us to purify SAG using the chromatography with or without DTT. Likewise, purified SAG is redox sensitive. Upon H2O2 expos ure, SAG forms oligomers as well as monomer doublets due to the formation o f the inter- or intramolecular disulfide bonds, respectively. This process can be reversed by DTT or prevented by pretreatment with the alkylating rea gent, N-ethylmaleimide (NEM). Although SAG contains two putative heme-bindi ng sites and a RING finger domain, the protein appears not to bind with hem e and to lack transcription factor activity as determined in a Gal4-fusion/ transactivation assay. Wildtype, but not RING finger domain-disrupted SAG m utants, prevents copper-induced lipid peroxidation. These results, along wi th our previous observations, suggest that SAG is an intracellular antioxid ant molecule that may act as a redox sensor to buffer oxidative-stress indu ced damage. (C) 1999 Elsevier Science Inc.