Expression of porcine leukocyte 12-lipoxygenase in a baculovirus/insect cell system and its characterization.
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.
Reddy
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A.B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Yoshimoto
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Yamamoto
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Funk
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CD
Marnett
L J
LJ
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006639:Histidine; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology*; D009036:Moths / Q000166:cytology; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives; D009942:Organometallic Compounds; D010455:Peptides / Q000235:genetics; D011233:Precipitin Tests; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D006893:Hydroxyeicosatetraenoic Acids; D009942:Organometallic Compounds; D010455:Peptides; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D016718:Arachidonic Acid; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001092:Arachidonate 12-Lipoxygenase; D009571:Nitrilotriacetic Acid