Cloning and expression of glutaredoxin from Xanthophyllomyces dendrorhous

• Main Authors: Shih,Jia-Ling, 施佳伶
• Other Authors: Lin, Chi-Tsai
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/24028367531889414202

碩士 === 國立臺灣海洋大學 === 生命科學暨生物科技學系 === 103 === Glutaredoxins (Grxs) are small thiol disulfide oxidoreductases. In bacterial,yeast and mammalian cells, Grxs appear to be involved in maintaining protein redox state. On the basis of their catalytic structure properties, Grxs can be classified into three categories. In this study, a cDNA encoding a putative Grx from Xanthophyllomyces dendrorhous was cloned by polymerase chain reaction (PCR). The DNA sequence has 487 bp, which would encode a Grx of 105 amino acids. The deduced protein showed high level of sequence homology when aligned with Grxs from other organisms, having a CPYC dithiol motif at the active site. To characterize the XdGrx, the coding region was subcloned into an expression vector pET-20b(+), which can be induced by IPTG, and transformed into E. coli BL21(DE3). The XdGrx is 11 kDa, and has 6 His tag. The expression of the Grx was purified by nickel chelating sepharose superflow column. Grx activity was determined by the β-hydroxyethyl (HED) disulfide assay.