Expression of porcine leukocyte 12-lipoxygenase in a baculovirus/insect cell system and its characterization.

Reddy RG, Yoshimoto T, Yamamoto S, Funk CD, Marnett LJ
Arch Biochem Biophys. 1994 312 (1): 219-26

PMID: 8031130 · DOI:10.1006/abbi.1994.1302

Arachidonate 12-lipoxygenase (12-LO) from porcine leukocytes was expressed in insect cells using a baculovirus expression vector. The recombinant 12-LO was expressed as an N-terminal fusion protein with a 31-amino acid polypeptide carrying a six-histidine tag and an enterokinase cleavage site. Maximal intracellular enzyme activity and protein levels were observed 48 h after infection of Spodoptera frugiperda cells with the recombinant virus. Cells were lysed and the recombinant protein was purified in a single step by Ni2+-nitrilotriacetate column chromatography. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. Recombinant enzyme catalyzed the formation of 12-hydroperoxy-5,8,10,14-eicosatetranoic acid and a small amount of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid. Chiral-phase HPLC analysis indicated that the 12-(S) enantiomer was the predominant product. The purified recombinant 12-lipoxygenase oxygenated linoleic acid to about 19% of the extent of oxygenation of arachidonic acid. Nordihydroguaiaretic acid and 5,8,11,14-eicosatetraynoic acid inhibited the recombinant enzyme with IC50's of 2.2 and 0.06 micM, respectively. Expression of cloned porcine leukocyte 12-LO in S. frugiperda cells and purification by Ni2+-nitrilotriacetate chromatography provides a straightforward method for isolation of milligram quantities of this form of 12-LO.

MeSH Terms (21)

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid Animals Arachidonate 12-Lipoxygenase Arachidonic Acid Baculoviridae Cells, Cultured Chromatography, Affinity Cloning, Molecular Histidine Hydroxyeicosatetraenoic Acids Kinetics Leukocytes Moths Nitrilotriacetic Acid Organometallic Compounds Peptides Precipitin Tests Protein Engineering Recombinant Fusion Proteins Substrate Specificity Swine

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