Overexpression, purification and characterization of human recombinant 15-lipoxygenase

摘要: Human 15-lipoxygenase was expressed to high levels (approx. 20% of cellular protein) in a baculovirus/insect cell expression system. Catalytically active enzyme readily purified (90–95% pure) from cytosolic fractions by anion-exchange chromatography on Mono Q column with approx. 95% recovery enzymatic activity. Routinely, yield 25–50 mg pure per L culture and specific activity 7.1–21 μmol 13-hydroxyoctadecadienoic acid (13-HODE)/mg·min (turnover rate 8.4–25 s−1) were obtained. Both the enzyme's iron content significantly increased addition ferrous ions either or insect medium during production. An isoelectric point 5.85 determined N-terminal amino sequence found be identical that predicted cDNA. The recombinant exhibits dual positional specificity arachidonic (formation 15S- 12S-hydroxyeicosatetraenoic (12S-HETE) ratio 12:1). Double oxygenation products 14R, various 8,15-DiHETE isomers also identified. With linoleic as substrate, pH-optimum 7.0 KM 3 μM determined. undergoes suicidal inactivation fatty oxygenation, is sensitive standard lipoxygenase inhibitors, oxygenates phospholipids, cholesterol esters, biomembranes human low-density lipoprotein. Contrary prior studies rabbit enzyme, no glycosylation detected.