|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1991 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1990 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Arachidonate 12-lipoxy, enase of porcine leukocytes was purified by immunoaffinity chromato, raphy using a monoclonal antibody against the enzyme. The purified enzyme was digested by lysyl endopeptidase which specifically cleaved the carboxyl side of a lysine residue. Amino acid sequences of the peptide fragments and of N-terminus of the enzyme were determined using a gas-phase sequencer. Two oligonucleotide probes were synthesized based upon the partial amino acid sequence of the peptides, and utilized as probes for screening. 12-Lipoxygenase CDNA was cloned from a CDNA library of porcine leukocytes. The cDNA of 12-lipoxygenase encoded 663 amino acids with a calculated molecular weight of 74, 911. Total RNA of various porcine tissues was separated by agarose gel electrophoresis, and subjected to Northern blot analysis. By far the largest amount of mRNA of the enzyme was detected in leukocytes, followed by pituitary, lung, jejunum and spleen, indicating that the enzyme was distributed in a variety of porcine tissues. In order to express the cDNA of 12-lipoxygenase in E. coli, an expression vector was constructed in a pKK plasmid with tac promoter. E. coli was transformed with the recombinant plasmid, and the 12-lipoxygenase protein was induced by the addition of isopropylthio-beta-D-galactoside. The extract of the transformed E. coli was incubated with arachidonic acid, and the reaction product was identified to be 12-hydroxy-eicosatetracnoic acid as assessed by reverse-phase HPLC. The result indicated that the expressed enzyme in E. coli had 12-lipoxygenase activity. Furthermore, Western blot analysis using anti-12-lipoxygenase antibody detected a positive band in the E. coli extract, confirming the expression of the enzyme protein.