Cloning, Expression and Characterization of Ipomoea batatas Glutaredoxin

• Main Authors: Xiao-Wen Chi, 祁孝雯
• Other Authors: Chi-Tsai Lin
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/94915820636899724439

碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 95 === The purpose of this thesis is to study antioxidant enzyme. A full length cDNA of 605 bp encoding a putative glutareoxin (Grx) from Ipomoea batatas was cloned by polymerase chain reaction (PCR). Nucleotide sequence analysis of this cDNA revealed that it comprised a complete open reading frame coding for 115 amino acid residues. To further characterize the Ipomoea batatas Grx, the coding region was subcloned into an expression vector pET-20b(+) and transformed into E.coli BL21 (DE3) pLySs. The expression of the Grx was purified by Ni2+ -nitrilotriacetic acid Sepharose superflow column. The purified enzyme then analyzed by 15 % SDS-PAGE. The enzyme retained 30 % activity after heating at 80 ℃ for 30 min. The enzyme activity was inhibited under acidic pH (pH 2.2). The enzyme retained 80 % activity under 0.4 M imidazole treatment. The enzyme showed 45 % activity after 2 h of incubation at 37 ℃ with trypsin. Furthermore, the enzyme was much resistant to proteolysis after 2 h of incubation at 37 ℃ with chymotrypsin.