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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48768

Title: 法夫酵母菌之抗氧化酵素2-Cys peroxiredoxin isozyme基因選殖、表現和特異性分析
Cloning and expression of 2-Cys peroxiredoxin isozyme from Xanthophyllomyces dendrorhous
Authors: Tz-You Shie
解資佑
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 法夫酵母菌;過氧化物還原酶;His-tag純化
Xanthophyllomyces dendrorhous;Peroxiredoxins;Expression
Date: 2016
Issue Date: 2018-08-22T06:31:08Z
Abstract: 過氧化物還原酶 (peroxiredoxins ; Prxs) 廣泛存在於細胞的許多胞器中,其酵素在對於保護生物免於活性氧的傷害及抗癌中有著重要的作用。Prxs利用cysteine(Cys) 殘基去催化使過氧化物的水平降低,基於可催化的Cys殘基數可被分為兩種形式 (1-Cys Prxs 和 2-Cys Prxs)。Prxs這個家族蛋白也根據它們的結構,及細胞內位置和作用模式等等被分為六型(Prxs1-6)。而2-Cys Prx又被分為兩種型態、典型Prx(Prx I-Prx IV),非典型Prx(Prx V)。本實驗比對不同物種之Prxs序列,找出保守區域的序列去設計專一性primers,再由法夫酵母(Xanthophyllomyces dendrorhous) 的cDNA庫中選殖出全長共940 bp之Prx cDNA,其蛋白轉譯區含756個鹼基對bp,可轉譯出252個胺基酸,序列比對之結果與2-Cys peroxiredoxins相似,故推斷其酵素為2-Cys Prx的isozyme (簡稱: Xd 2C-Prx Iso)。其預測分子量為28.2 kDa。以人類之Prx (PDB ID: 2Z9S)為模板,模擬蛋白3-D結構,同時辨識Prxs所含的兩個Cys活性催化位(Cys 106與229)。進一步將Xd 2C-Prx Iso的表現區選殖入至表現載體pET-20b(+),並且轉形至大腸桿菌C43 (DE3) 和BL21(DE3),將轉型之大腸桿菌培養於20 mL LB broth中並加入IPTG誘導表現重組蛋白(IPTG最終濃度為0.5 mM),利用鎳螯合親和性管柱層可純化出26 kDa之Xd 2C-Prx Iso之可溶性蛋白。以Ferrithiocyanate assay進行可溶性之Xd 2C-Prx Iso活性測定。
Peroxiredoxins (Prxs) play important roles in antioxidation and anticancer . Prxs are classified into two types, 1-Cys or 2-Cys, based on whether they contain one or two conserved Cys residues. Based on its structure, the Prxs were classified into six types: four 2-Cys Prxs (Prxs I–IV), one atypical 2-Cys Prxs (Prx V), and one 1-Cys Prx isoform (Prx VI). In this study, full-length cDNA of 940 bp encoding the putative Xd 2C-Prx Iso from. Xanthophyllomyces dendrorhous. was cloned by PCR. The coding region of Xd 2C-Prx Iso encodes 252 amino acid residues with a calculated molecular mass of 28.2 kDa. A 3-D structural model of Xd 2C- Prx Iso was predicted based on the crystal structure of Homo sapiens Prxs (PDB ID: 2Z9S). The active site of Xd 2C-Prx Iso were predicted as Cys 106 and Cys 229. The coding region of Xd 2C-Prx Iso cDNA from X. dendrorhous was subcloned into an expression vector, PET-20b(+), and then transformed into E coli C43 (DE3) and E coli BL21 (DE3). The transformed E coli C43 (DE3) and Escherichia coli BL21 (DE3) containing the Xd 2C-Prx Iso was grown in 20 ml of Luria Bertani (LB) medium. Protein expression was induced by addition of isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM, and purified by His-tag technique. The soluble recombinant Xd 2C-Prx Iso was obtained in Escherichia coli C43 (DE3) and Escherichia coli BL21 (DE3). Protein activity was analyzed by us Ferrithiocyanate assay.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010336023.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48768
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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