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Saccharomyces cerevisiae에서 GAL 또는 GAP 프로모터 조절에 의한 재조합 Inulinase의 발현 및 분비
남수완,임현정,정봉현,장용근 동의대학교 기초과학연구소 1997 基礎科學硏究論文集 Vol.7 No.1
To investigate the promoter effect on heterologous gene expression in S. cerevisiae, the recombinant plasmids pYI11, pYI12, pYI10-2, and pYIGP was constructed to contain the inulinase gene (INU1) as a reporter under the control of GAL10, GAL7, GAL1 and GAP promoters, respectively. When the yeasts transformants were cultivated on galactose-containing rich media, the cell growth reached to 36-39 OD_600 at 72 hours of cultivation. The specific growth rates of the cells harboring the four different plasmids decreased similarily : they dropped from 0.24 h^-1 during the glucose-consuming period to 0.04-0.10 h^-1 during the galactose-consuming period (gene expression phase for GAL promoter system). After the depletion of glucose, the expression of inulinase gene was started and reached to maximal levels of 4.3(GAL1 promoter), 4.0(GAL10 promoter), 3.8(GAL7 promoter), and 1.6(GAP promoter) unit/mL at 72 hours of cultivation. Based on the maximal expression level and activity staining on the plate, the promoter strength was in the order of GAL1, GAL10, GAL7, and GAP promoter. While the GAL-promoter systems showed a high plasmid stabilities of more than 78%, the GAP-promoter plasmid revealed a lower plasmid stability of 55%. Most of inulinase activity (98%) was found in the extracellular medium, indication that the secretion efficiency of inulinase is independent on the type of promoter.
인간 복막 중피 세포의 Transforming Growth Factor-β1(TGF-β1) 합성에 관한 연구
한대석,최진희,윤견일,강덕희,임현정,홍영숙 대한신장학회 1999 Kidney Research and Clinical Practice Vol.18 No.3
Objective:to investigate the effect of high glucose and spent peritoneal dialysate on the TGF-β1 synthesis of cultured human peritoneal MC(HPMC); to examine the effect of costimulation with high glucose or dialysate and cytokines, interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α), on transforming growth factor(TGF-β1) synthesis of HPMC. Design:HPMCs were exposed to different concentrations of glucose(30, 60 & 90 mM/L) or spent peritoneal dialysate for 48 hours in the absence or presence of IL-1β(1ng/ml) and TNF-α(1ng/ml). TGF-β1 mRNA expression was assessed by Northern blot analysis and TGF-β1 protein synthesis and release by Western blot analysis with immunoprecipitation. Results:Exposure of MC to high glucose condition(30mM, 60mM & 90mM of D- glucose) induced 2.3-, 3.6- and 4.0-fold increases in TGF-β1 mRNA expression of MC with enhanced TGF-β1 protein synthesis and secretion into the media. Incubation with spent dialysate also significantly increased TGF-β1 mRNA expression & protein pared to control media(P$lt;0.05) Stimulation with IL-1β(1ng/ml) or TNF-α(1ng/ml) significantly increased TGF-β1 mRNA expression after 48 hours above the control level by 2.7-fold and 2.1-fold, respectively. However, TNF-α-induced increase in TGF-β1 mRNA expression was not translated into TGF-β1 protein secretion whereas IL-1β stimulation induced a significant increase in TGF-β1 protein secretion as well as TGF-β1 mRNA expression. Combined stimulation of high glucose or spent dialysate together with IL-1β or TNF-α showed a greater increase in TGF-β1 mRNA expression and protein secretion compared to stimulation with high glucose or spent dialysate alone. Conclusion:Our results clearly show that high glucose concentration of peritoneal dialysate and spent dialysate themselves might be sufficient to stimulate the production of TGF-β1 by peritoneal mesothelial cell. This state of chronic induction of TGF-β1 is further exaggerated in the presence of peritonitis because of stimulatory effect of proinflines, resulting in the augmented TGF-β1 synthesis, thus promoting peritoneal fibrosis.