| Co-Investigator(Kenkyū-buntansha) |
YAMAGATA Hideo School of Life Science,Tokyo University of Pharmacy and Life Science,Professor.., 生命科学部, 教授 (20023468)
KOJIMA Masaki School of Life Science,Tokyo University of Pharmacy and Life Science,Assistant., 生命科学部, 助手 (90277252)
INOUE Hideshi School of Life Science,Tokyo University of Pharmacy and Life Science,Assistant Professor., 生命科学部, 助教授 (20184765)
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| Research Abstract |
Full-length cDNAs of two aspartic proteases and a partial-length CDNA of one aspartic protease were isolated from the CDNA libraries of Brugia malayi and their base sequences were determined and amino acid sequences deduced. Analysis of a phylogenetic tree indicated that the two major aspartic proteteases from Brugia malayi belong to different groups ofaspartic proteases. Histochemical analysis using their antibodies showed that one of them is present only in the esophagus and intestines while the other is present in various tissues and Organs including the esophagus, intestines, body wall and reproductive organs. These results suggested that the former is involved in the gastro-intestinal digestion of nutrient proteins while the other is responsible for ihtracellular protein degradation. Three full-length cDNAs. and four partial-length cDNDs of cysteine proteases, one full-length CDNA and four partial-length cDNAs of metalloproteases, and two cDNAs of deubiquitinating enzymes were als
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o isolated, and their sequences were determined to deduce the corresponding amino acid sequences. The full-length CDNA for a metalloprotease coded for a sequence sinmilar to that of the non-catalytic subunit of mitochondrial processing protease. Heterologous expression of some of these proteases were, attempted using Escherichial coli and Bacillus brevis expression systems. In the E. coli system, aconsiderable amount of the proteases was produced, but as an inactive inclusion bodies. The refolding of the denatured enzymes to potentially active enzymes, however, was found to be rather difficult, despites of a number of attempts under different conditions. The B. brebis system also-failed-to give a reasonable amount of the potentially active enzymes. Further elaborate studies are necessary in this regard. Along with these studies, a comparative studies were carried out with several proteases Caenorhabditis elegans, a non-parasitic nematode, using CDNA cloning and RNA interference, and several important proteases were identified. Less
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