Expression of porcine leukocyte 12-lipoxygenase in a baculovirus/insect cell system and its characterization. 1994

R G Reddy, and T Yoshimoto, and S Yamamoto, and C D Funk, and L J Marnett
A.B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.

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.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D007962 Leukocytes White blood cells. These include granular leukocytes (BASOPHILS; EOSINOPHILS; and NEUTROPHILS) as well as non-granular leukocytes (LYMPHOCYTES and MONOCYTES). Blood Cells, White,Blood Corpuscles, White,White Blood Cells,White Blood Corpuscles,Blood Cell, White,Blood Corpuscle, White,Corpuscle, White Blood,Corpuscles, White Blood,Leukocyte,White Blood Cell,White Blood Corpuscle
D009036 Moths Insects of the suborder Heterocera of the order LEPIDOPTERA. Antheraea,Giant Silkmoths,Giant Silkworms,Silkmoths, Giant,Silkworms, Giant,Antheraeas,Giant Silkmoth,Giant Silkworm,Moth,Silkmoth, Giant,Silkworm, Giant
D009571 Nitrilotriacetic Acid A derivative of acetic acid, N(CH2COOH)3. It is a complexing (sequestering) agent that forms stable complexes with Zn2+. (From Miall's Dictionary of Chemistry, 5th ed.) Aluminum Nitrilotriacetate,Dysprosium Nitrilotriacetate,Trisodium Nitrilotriacetate,Acid, Nitrilotriacetic,Nitrilotriacetate, Aluminum,Nitrilotriacetate, Dysprosium,Nitrilotriacetate, Trisodium
D009942 Organometallic Compounds A class of compounds of the type R-M, where a C atom is joined directly to any other element except H, C, N, O, F, Cl, Br, I, or At. (Grant & Hackh's Chemical Dictionary, 5th ed) Metallo-Organic Compound,Metallo-Organic Compounds,Metalloorganic Compound,Organometallic Compound,Metalloorganic Compounds,Compound, Metallo-Organic,Compound, Metalloorganic,Compound, Organometallic,Compounds, Metallo-Organic,Compounds, Metalloorganic,Compounds, Organometallic,Metallo Organic Compound,Metallo Organic Compounds
D010455 Peptides Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are considered to be larger versions of peptides that can form into complex structures such as ENZYMES and RECEPTORS. Peptide,Polypeptide,Polypeptides
D011233 Precipitin Tests Serologic tests in which a positive reaction manifested by visible CHEMICAL PRECIPITATION occurs when a soluble ANTIGEN reacts with its precipitins, i.e., ANTIBODIES that can form a precipitate. Precipitin Test,Test, Precipitin,Tests, Precipitin
D011993 Recombinant Fusion Proteins Recombinant proteins produced by the GENETIC TRANSLATION of fused genes formed by the combination of NUCLEIC ACID REGULATORY SEQUENCES of one or more genes with the protein coding sequences of one or more genes. Fusion Proteins, Recombinant,Recombinant Chimeric Protein,Recombinant Fusion Protein,Recombinant Hybrid Protein,Chimeric Proteins, Recombinant,Hybrid Proteins, Recombinant,Recombinant Chimeric Proteins,Recombinant Hybrid Proteins,Chimeric Protein, Recombinant,Fusion Protein, Recombinant,Hybrid Protein, Recombinant,Protein, Recombinant Chimeric,Protein, Recombinant Fusion,Protein, Recombinant Hybrid,Proteins, Recombinant Chimeric,Proteins, Recombinant Fusion,Proteins, Recombinant Hybrid
D002478 Cells, Cultured Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others. Cultured Cells,Cell, Cultured,Cultured Cell
D002846 Chromatography, Affinity A chromatographic technique that utilizes the ability of biological molecules, often ANTIBODIES, to bind to certain ligands specifically and reversibly. It is used in protein biochemistry. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Chromatography, Bioaffinity,Immunochromatography,Affinity Chromatography,Bioaffinity Chromatography

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