Cloning and characterization of glutaredoxin from Chlorella sorokiniana T89

• Main Authors: Chu-Ying Cheng, 鄭竹螢
• Other Authors: 蕭介夫
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/c24r6m

碩士 === 國立中興大學 === 食品暨應用生物科技學系所 === 100 === Glutaredoxins (Grxs) are ubiquitous oxidoreductases in aerobic organism. They catalyze glutathione-dependent reactions to reduce protein disulfide linkage caused by oxidative stress. A full length cDNA encoding a glutaredoxin gene with 321 nucleotides was isolated from Chlorella sorokiniana T89. Nucleotides sequence analysis showed that it deduced 106 amino acid residues, the molecular weight of protein is predicted approximately 11.6 kDa. The deduced amino acid sequence is conserved among the reported dithiol Grxs because the characteristic catalytic motif C25-P-Y-C28. Recombinant glutaredoxin was subcloned into an vector pET-20b(+) and expressed in Escherichia coli BL21(DE3) as a C-terminal histidine-tagged fusion protein. The expression of glutaredoxin was purified by TALON affinity chromatography and analyzed by 16 % tricine SDS-PAGE. glutaredoxin activity was assayed by β-hydroxyethyl disulfide (HED) assay. The optimal pH for the recombinant glutaredoxin was 8.5, and the optimal temperature was 50 C. The recombinant glutaredoxin exhibition a good thermal stability and pH stability (pH 3~11). The Michaelis constant (Km) values for HED was 0.17 mM. The enzyme activity was significantly inhibited by 1 mM Cu2+, Zn2+, and Fe3+. The enzyme was susceptible inactivation in iodoacetamide, SDS, and urea.