Cloning and expression of 2-Cys peroxiredoxin isozyme from Xanthophyllomyces dendrorhous

• Main Authors: Tz-You Shie, 解資佑
• Other Authors: Lin, Chi-Tsai
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/37725047900398973017

碩士 === 國立臺灣海洋大學 === 生命科學暨生物科技學系 === 104 === 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.