Browsing by Author "Lin, Chih Sheng"

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Construction and expression of rabbit neutrophil peptide-1 gene in Escherichia coli
(2010-06-01)
Sun, Yu Ling
;
Kuan, Tang Ching
;
Lin, Yi Juain
;
Chou, Yu Chi
;
Lin, Chih Sheng
Rabbit neutrophil peptide-1 (NP-1) is a prototypic rabbit alpha-defensin with a broad antimicrobial spectrum. The coding sequence of NP-1 was amplified and cloned into pET-31b(+) to construct an expression vector, pET31-NP1, which was transformed into E. coli BLR(DE3) pLysS for expressing the fusion NP-1 protein (fNP-1). The fNP-1 is downstream of a ketosteroid isomerase (KSI) and upstream of a (His)(6)-Tag, as KSI-NP1-His(6). The optimal condition, cultivation in enriched LB medium and induction with 0.5 mM IPTG for 6 h, was determined for fNP-1 production. The fNP-1 was purified by Ni-NTA resin and cleaved by cyanogen bromide to release matured NP-1 peptide. The matured NP-1 peptide showed significant antimicrobial activities against clinical bacteria, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. The application of this expression approach represents a potential method to produce NP-1 by fusion protein expressed in E. coli without cytotoxicity.
Identifying the Regulative Role of NF-kappa B Binding Sites Within Promoter Region of Human Matrix Metalloproteinase 9 (mmp-9) by TNF-alpha Induction
(2013-01-01)
Wu, Hsi Tien
;
Sie, Syu Sheng
;
Kuan, Tang Ching
;
Lin, Chih Sheng
;
生物科技學系
Matrix metalloproteinase 9 (MMP-9), a member of MMP family, is involved in many physiological processes, including cardiovascular disease (CVD). Tumor necrosis factor-alpha (TNF-alpha) is considered a cytokine with pleiotropic biological capabilities and leads to the process of CVD when TNF-alpha is abnormally released and stimulates MMP-9 expression and activation. In this study, we investigated the molecular mechanism of TNF-alpha-regulated MMP-9 expression. The experimental results confirm that TNF-alpha could upregulate MMP-9 expression in heart myoblast H9c2 cells of rat. To evaluate the MMP-9 regulation at transcriptional level, a DNA fragment of 2.2 kb (-2168/+18) of human mmp-9 promoter region was cloned and constructed in a vector of luciferase reporter gene. The 2.2-kb sequences were identified as having three candidate nuclear factor-kappa B (NF-kappa B) binding sites: NF-kappa B I (-1418/-1409), NF-kappa B II (-626/-617), and NF-kappa B III (-353/-345). A series of reporter vectors with the mutated NF-kappa B sites of mmp-9 promoter sequences were constructed and transfected into H9c2 cells. The results show that the NF-kappa B II binding site (-626/-617) within the promoter region of mmp-9 plays a key role in upregulation of mmp-9 expression by TNF-alpha induction. In addition, we also first identified that the NF-kappa B I, similar to c-Rel, might be one of the NF-kappa B families to regulate mmp-9 expression.