Monothiol glutaredoxin from Taiwanofungus camphoratus: cloning, expression and properties

• Main Authors: I-Ching Chen, 陳怡靜
• Other Authors: Chi-Tsai Lin
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/74395067118580894220

碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 97 === The purpose of this thesis is to study an antioxidant enzyme, glutaredoxins (Grxs) from Taiwanofungus camphoratus. Grxs are small thiol disulfide oxidoreductases. In bacterial, yeast and mammalian cells, Grxs appear to be involved in maintaining protein redox state. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs, monoGrxs) has been identified in prokaryotes and eukaryotes but not well characterized. A cDNA encoding monoGrx from Taiwanofungus camphoratus fruit body (TcmonoGrx) was cloned by polymerase chain reaction (PCR). The DNA sequence has 1037 bp, which would encode a monoGrx of 238 amino acids with a molecular mass of 27 kDa. The deduced amino acid sequences is well-conserved among the reported monoGrxs, Trx-Grx class especially, including two domains : thioredoxin (Trx)-like domain (WAD/EPCK) and Grx domain (CGFS). To further characterize the TcmonoGrx, the coding region was subcloned into an expression vector pET-20b(+) and transformed into Escherichia coli. The expression of the TcmonoGrx was purified by nickel chelating sepharose superflow column. Analysis of TcmonoGrx activity, it was inactive as a glutathione (GSH)-disulfide oxidoreductase in a standard Grx assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and GSH reductase. An engineered CGFC168 active site of Grx domain mutant did not gain activity either. In GSSG (oxidized GSH) reduction and DTNB assay, TcmonoGrx was demonstrated to reduce GSSG to GSH by directly using NADPH, and the reduction corresponding to the consumption of one molecule of NADPH per molecule of GSSG. In analysis of mutants activity, TcmonoGrx was proved to use cysteine to reduce GSSG, and Grx domain was the major active site. The Michaelis constant (Km) values for NADPH and GSSG were 0.041 and 0.064 mM for the TcmonoGrx. The protein’s half-life of deactivation at 60 ℃ was 10.5 min. The enzyme activity was stable in a broad pH range from 4.0 to 10.0. The enzyme activity lost 40 % in the presence of 0.2 M or up to 0.8 M imidazole. Last, the enzyme showed 70 % and 50 % activity after 40 min of incubation at 37 ℃ with chymotrypsin and trypsin, respectively.