法夫酵母2-Cys peroxiredoxin基因選殖及表現之研究

Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48765

Title:	法夫酵母2-Cys peroxiredoxin基因選殖及表現之研究 Cloning and expression of 2-Cys peroxiredoxin from Xanthophyllomyces dendrorhous
Authors:	Hsiao, Ching-Ta 蕭敬達
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;過氧化氫酶 Xanthophyllomyces dendrorhous;peroxiredoxin;expression
Date:	2016
Issue Date:	2018-08-22T06:31:07Z
Abstract:	Peroxiredoxin是ㄧ類過氧化酶 (peroxidase) ，屬於抗氧化蛋白，廣泛存在於原核和真核生物中。Prx蛋白的N-端都具有保守的Cys殘基，依據Cys殘基的數目以及保守性分為1-Cys Prx和2-Cys Prx。Peroxiredoxin的目的是將過氧化氫還原成氧化氫，可見Prx是一個很重要的抗氧化酵素。以不同物種來設計Prx之胺基酸序列保守區域設計引子，再利用聚合酶連鎖反應，從法夫酵母菌cDNA庫中選殖出2-Cys peroxiredoxin (簡稱XdPrx) 之全長 cDNA序列，全長大小為 942 bp，而轉譯區為 600 bp，可以譯出 199 個胺基酸，預測分子質量約為22 kDa。XdPrx 以pET-20b(+) 作為表現載體，以E.coli C43 (DE3) 作為表現宿主，經由1mM IPTG 誘導20 ml 的培養液，即可表現出XdPrx。以含有鎳螯合物凝膠 (nickel chelating sepharose) 之親合性管柱進行親合型結合來純化目標蛋白，其分子質量約為 50 kDa 推測為雙倍體。之後以ferrithiocyanate assay測定其活性，測得的蛋白活性不穩定，在 60℃下熱失活常數 (kd) 為 9.62x10-2 min-1，半衰期約為3 min。 One type of peroxidases was peroxiredoxin, exist in prokaryotes and eukaryotes. Peroxiredoxin has conservative cysteine residue at the N-terminal portion. Peroxiredoxin provided the protective antioxidant by reducing hydrogen peroxide to H2O. Peroxiredoxins play important roles in antioxidation. cDNA of 942 base pairs containing a complete open reading coding 198 amino acid residues encoding a putative Prx (named XdPrx) was cloned from Xanthophyllomyces dendrorhous. The molecular mass was predicted 22 kDa. The deduced protein showed high level of sequence homology when aligned with Prx from other organisms. The coding region of XdPrx cDNA from Xanthophyllomyces dendrorhous was introduced into an expression vector, pET-20b(+), and transformed into Escbericbia coli CK3(DE3). The recombinant 6His-tag XdPrx was expressed and purified by Ni2+-nitrilotriacetic acid Sepharose column. The enzyme has a half-life of 3 min at 60 °C and kd value is 9.62x10-2 min-1.
URI:	http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010336022.id http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48765
Appears in Collections:	[生命科學暨生物科技學系] 博碩士論文

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