Heterologous expression of dehydroascorbate reductase from rice and its application to determination of dehydroascorbate concentrations.
We constructed an expression vector for rice dehydroascorbate reductase (DHAR) (EC 1.8.5.4) with a polyhistidine tag at the amino terminus and introduced the vector into several strains of Escherichia coli. On conventional induction treatment with isopropylthiol-beta-D-galactoside, E. coli harboring rice DHAR cDNA produced doublet polypeptides of about 27 kDa. Induction duration or growth temperature did not affect the ratio of these polypeptides. Only the larger polypeptide, corresponding to full-length recombinant DHAR, was produced in E. coli supplemented with tRNAs for several minor codons. Most of the enzymatic characteristics of the recombinant DHAR were similar to those of the native one, although the recombinant protein showed increased heat susceptibility. Using recombinant DHAR, we developed a method for simple and precise determination of dehydroascorbate concentrations in tissue extracts by spectrophotometry, and we successfully applied the method to several fruit juices and vegetables.
Amako
Katsumi
K
Faculty of Nutrition, Kobe Gakuin University, Japan. amakok@nutr.kobegakuin.ac.jp
Ushimaru
Takashi
T
Ishikawa
Ayami
A
Ogishi
Yasuka
Y
Kishimoto
Ritsuko
R
Goda
Kiyoshi
K
D001628:Beverages; D003683:Dehydroascorbic Acid / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005638:Fruit / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014675:Vegetables / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D010088:Oxidoreductases; C020666:glutathione dehydrogenase (ascorbate); D003683:Dehydroascorbic Acid