| Co-Investigator(Kenkyū-buntansha) |
KAWABE Yosiki Chugai Pharmaceutical Co., LTD., Senior Scientist., 創薬研究所, 研究員
HAMAKUBO Takao Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Associate Professor., 先端科学技術研究センター, 助教授 (90198797)
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| Research Abstract |
We have been reported that SREBP-2 (sterol regulatory element-binding protein-2) play a key role in the regulation of cholesterol butt not fatty acid metabolism by establishing cell lines in which human SREBP-2 could be induced by IPTG.We also constructed a sensitive assay method for detection of SREBP cutting enzymes using internally quenched fluorogenic peptide substrate, the amino acid sequence of which is the same as the predicted cutting site. By this assay system, we purified neprilysin and cathepsin B as cleaving enzymes in hamster liver microsome fraction. These enzymes were considered to be degradative enzymes for SREBPs in endoplasmic reticulum. The soluble part of human site 1 protease (S1P) was expressed in CHO cells and purified with his-tag affinity column. As the activity of soluble S1P was hardly detectable by the above assay method, we developed a new expression system using the baculovirus. By the baculovirus expression system, we have succeeded to express whole S1P protein and detected Ca^<++> dependent enzymatic activities primarily on S1P expressed budded viruses. We have also detected the expression of membrane proteins such as SREBP-2, SCAP (SREBP cleavage activating protein) and HMG-CoA reductase in viral fractions of culture supernatant of transfected Sf9 cells. This expression system is considered to be a useful tool for expressing membrane proteins. On the other hand, we analyzed genomic structures of S1P, SCAP, and HMG-CoA reductase, and found that exon-shuffling may have occurred in evolutionally step in those proteins that have sterol sensing domain (SSD).
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