title: Intra- and extracellular expression of rabbit reticulocyte 15-lipoxygenase in the Baculovirus/insect cell system.
abstract: Rabbit reticulocyte 15-lipoxygenase was expressed as intracellular enzyme and as export protein in High Five cells. While intracellular expression in the baculovirus/insect cell system was already reported for various mammalian lipoxygenases, we have developed a strategy for secretion of this cytosolic enzyme using the signal sequence of human interleukin-2. Expression levels of 10 mg/liter (intracellular strategy) and 18 mg/liter (extracellular strategy) were obtained. The recombinant enzyme expressed as intracellular protein was purified to apparent homogeneity by anion-exchange chromatography on a Mono-Q column with an overall recovery of 80% enzyme activity. For the final enzyme preparation, a specific linoleic acid oxygenase activity of 16.7 micromol 13-hydroperoxyoctadeca-9,11-dieic acid formation mg-1 min-1 was determined, which corresponds to a molecular turnover number of 21 s-1. Similar turnover rates have been reported for the native rabbit 15-lipoxygenase. Extracellularly expressed recombinant 15-lipoxygenase exhibited a heterogeneity in anion-exchange chromatography. Three major peaks of 15-lipoxygenase activity were eluted from a Mono-Q column and the relative amounts of these isoforms varied from batch to batch of enzyme expression. One of the major isoenzymes which cochromatographed with the native 15-lipoxygenase was purified to homogeneity from the cell-free culture supernatant and exhibited a specific activity of 5.1 micromol 13-hydroperoxyoctadeca-9,11-dienoic acid formation mg-1 min-1 (turnover rate of 6.1 s-1). The recombinant enzyme species were characterized with respect to their protein-chemical and enzymatic properties and no differences to the native rabbit 15-lipoxygenase were detected.
authors.affiliation: Institute of Biochemistry, University Clinics (Charité), Humboldt University, Hessische Strasse 3-4, 10115, Berlin.
authors.forename: S
authors.initials: S
authors.lastname: Borngräber
authors.forename: E
authors.initials: E
authors.lastname: Grabenhorst
authors.forename: M
authors.initials: M
authors.lastname: Anton
authors.forename: H
authors.initials: H
authors.lastname: Conradt
authors.forename: H
authors.initials: H
authors.lastname: Kühn
mesh_terms: D000818:Animals; D001093:Arachidonate 15-Lipoxygenase / Q000737:chemistry*; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000235:genetics; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis; D008563:Membrane Lipids / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012156:Reticulocytes / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D006893:Hydroxyeicosatetraenoic Acids; D008563:Membrane Lipids; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D001093:Arachidonate 15-Lipoxygenase