Expression of Modified Human Cytochrome P450 3A4 in Escherichia coli and Purification and Reconstitution of the Enzyme

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

“…There are several reports that demonstrate activation of CYP3A4-dependent reactions by GSH in reconstituted systems in vitro [44][45][46]. Since the maximal activation is observed immediately after addition of GSH, GSH-dependent reduction of protein thiols does not appear to be involved [44,46].…”

“…Since the maximal activation is observed immediately after addition of GSH, GSH-dependent reduction of protein thiols does not appear to be involved [44,46]. The effect of GSH, which apparently involves a strengthening of the interactions of CYP3A4 with NADPH-cytochrome P450 reductase (CPR) [46], is amplified considerably in the presence of cytochrome b 5 [46] and increased at high ionic strength [45].…”

Bacterial expression of two human aryl sulfotransferases

“…Similarly, no visible difference was observed between the subcellular localisation of expressed proteins. While N-terminal modifications have been shown to be important in enhancing P450 expression in E. coli, particular modifications have failed to produce consistent effects [8]. The present study involved only a single mutation at the second position and it is possible that further mutations in this region may alter expression levels.…”

“…Protein expression was induced with isopropyl-β--thiogalactopyranoside (IPTG). Cells were harvested after 24 h, treated with lysozyme and sonicated, and subcellular fractions were prepared by differential centrifugation as described previously [8].…”

“…Bacteria were cultured for 24 h at 30°C with 1 mM IPTG and subcellular fractions were prepared as described previously [8]. Lanes 1 and 2, 50 μg of total protein from COS cells transfected with pCMV5/HAST1 and pCMV5/HAST3, respectively [2,3].…”