Cloning, Expression and Activity Modulation of Human Squalene Synthase

• Main Authors: HUANG - CHI CHEN, 陳璜琪
• Other Authors: Chun-Hung Chiu
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/77762162428123053648

碩士 === 弘光科技大學 === 生物科技研究所 === 94 === ABSTRACT Squalene, a naturally occurring hydrocarbon triterpene type containing six isoprene units, is an important intermediate in the endogenous biosynthesis of cholesterol. The beneficial effects of squalene on health are accumulating and primarily attributed to its impact on the cholesterol metabolism. Recently, one working hypothesis has been proposed that squalene may be a key factor in the cancer-risk reducing effect of olive oil. Although epidemiological and animal studies suggest anti-cancer properties of squalene, to date no human trials have been conducted. It could be due to the lacking of clear mechanisms. The formation of squalene is catalyzed by squalene synthase (SQS), the first enzyme committed exclusively to sterol synthesis. Studies with mammalian cells have shown that regulation of SQS is crucial for balancing the sterol and nonsterol isoprene synthesis in response to changing cellular requirements. In connection with our interest in the disease prevention efficacy of squalene and its possible mechanisms, we sought a reliable source of the catalyzed enzyme, squalene synthase. Therefore, the overall objective of this study was designed to clone and construct a cDNA for human squalene syntase (hSQS) for future examination on functions and modulations of squalene and even cholesterol biosynthesis. In this study, total RNA was extracted from human hepatic carcinoma cell (HepG2), followed by RT-PCR to amplify the targeted DNA, which is about 1.2 kb. The desired gene was then subcloned to pQE9 and transformed to E coli (JM109) and the purified DNA was sequenced and compared with the published data. For enzyme activity analysis. In the recombinant expression plasmid was transformed to JM15 and the squalene synthase protein was amplified under IPTG induction. The enzyme activity was determined after Ni-NTA purification and using [3H] FPP as substrate. Since squalene synthase is the first enzyme in the cholesterol biosynthesis pathway, modulation of this enzyme may further regulate cholesterol formation and this may serve as the basis for nutraceutical and pharmaceutical design. Accordingly, we examined the effect of the hawthorn, ellagic acid and lovastatin, all have been demonstrated having the potential to reduce cholesterol, on squalene synthase activity. The results showed hawthorn and ellagic acid inhibited the squalene synthase activity with dose-dependent and on the other hand, lovastatin activated the squalene synthase even it inhibited HMG-CoA reductase. Keyword：squalene、cholesterol、squalene synthase