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 si x-histidine tag apposed to the human platelet 12-lipoxygenase cDNA, un der control of the polyhedrin promoter, was constructed. Recombinant 1 2-lipoxygenase baculoviruses were used to infect Spodoptera frugiperda insect cells (Sf9). At 54 h post-infection, maximal 12-lipoxygenase a ctivity and protein levels were achieved; the enzyme was purified to a pparent homogeneity in a single step by nickel-ion-chelation chromatog raphy in which the (His)6-tagged 12-lipoxygenase was eluted with 100 m M imidazole. The purified enzyme metabolized arachidonic acid almost e xclusively to 12-hydroperoxyeicosatetraenoic acid with little, if any, epoxyalcohol or reduction products and had a V(max) of 2-4 mumol min- 1 mg protein-1, K(m) of 10 muM and k(cat) of almost-equal-to 250 min-1 . Linoleic acid, on the other hand, was converted to (13S)-13-hydroper oxy-octadecadienoic acid at a rate which was about 2% of that obtained with arachidonic acid as substrate, but displayed the same K(m). The enzyme was most active between pH 7.5-8 and activity was stimulated si gnificantly in the presence of 0.006% Tween-20. A polyclonal antibody to the recombinant enzyme was generated and found to recognize a singl e 75-kDa band in platelets, human erythroleukemia cells and 12-lipoxyg enase baculoviral-infected Sf9 cells by immunoblot and immunoprecipita tion methods. 12-Lipoxygenase protein represented 0.1% of the total so luble protein in platelet preparations. In immunofluorescence experime nts 12-lipoxygenase was observed in the cytoplasm of infected insect c ells and in the human megakaryoblastic DAMI cell line. The isolation o f large quantities of pure human platelet 12-lipoxygenase should facil itate detailed biochemical structure/function studies.