Purification and characterization of recombinant histidine‐tagged human platelet 12‐lipoxygenase expressed in a baculovirus/insect cell system

Abstract

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 μmol min−1 mg protein−1, Km of 10 μM and kcat of ∼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. Copyright © 1993, Wiley Blackwell. All rights reserved