Purification and characterization of recombinant histidine-tagged human platelet 12-lipoxygenase expressed in a baculovirus/insect cell system.
A baculoviral expression vector consisting of a sequence encoding a six-histidine tag apposed to the human platelet 12-lipoxygenase cDNA, under control of the polyhedrin promoter, was constructed. Recombinant 12-lipoxygenase baculoviruses were used to infect Spodoptera frugiperda insect cells (Sf9). At 54 h post-infection, maximal 12-lipoxygenase activity and protein levels were achieved; the enzyme was purified to apparent homogeneity in a single step by nickel-ion-chelation chromatography in which the (His)6-tagged 12-lipoxygenase was eluted with 100 mM imidazole. The purified enzyme metabolized arachidonic acid almost exclusively to 12-hydroperoxyeicosatetraenoic acid with little, if any, epoxyalcohol or reduction products and had a Vmax of 2-4 mumol min-1 mg protein-1, Km of 10 microM and kcat of approximately 250 min-1. linoleic acid, on the other hand, was converted to (13S)-13-hydroperoxy-octadecadienoic acid at a rate which was about 2% of that obtained with arachidonic acid as substrate, but displayed the same Km. The enzyme was most active between pH 7.5-8 and activity was stimulated significantly in the presence of 0.006% Tween-20. A polyclonal antibody to the recombinant enzyme was generated and found to recognize a single 75-kDa band in platelets, human erythroleukemia cells and 12-lipoxygenase baculoviral-infected Sf9 cells by immunoblot and immunoprecipitation methods. 12-Lipoxygenase protein represented 0.1% of the total soluble protein in platelet preparations. In immunofluorescence experiments 12-lipoxygenase was observed in the cytoplasm of infected insect cells and in the human megakaryoblastic DAMI cell line. The isolation of large quantities of pure human platelet 12-lipoxygenase should facilitate detailed biochemical structure/function studies.
Chen
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Department of Pharmacology, Vanderbilt University, Nashville, TN 37232.
Brash
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Funk
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D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D016718:Arachidonic Acid / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001792:Blood Platelets / Q000201:enzymology*; D002451:Cell Compartmentation; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D003429:Cross Reactions; D003593:Cytoplasm / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D006639:Histidine; D006801:Humans; D015289:Leukotrienes / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013379:Substrate Specificity
D015289:Leukotrienes; D010455:Peptides; D011994:Recombinant Proteins; C033223:polyhistidine; D016718:Arachidonic Acid; D006639:Histidine; C021201:12-HPETE; D001092:Arachidonate 12-Lipoxygenase