Purification and characterization of two ascorbate peroxidases of rice (Oryza sativa L.) expressed in Escherichia coli.
To clarify the diversity and function of isozymes of ascorbate peroxidase (APX) in plants, a method of producing large quantities of these proteins is needed. Here, we describe an Escherichia coli expression system for the rapid and economic expression of two rice APX genes, APXa and APXb (GeneBank accession Nos. D45423 and AB053297, respectively). The two genes were cloned into the pGEX-6p-3 vector to allow expression of APX as a glutathione-S-transferase (GST) fusion protein. The GST-APXa and GST-APXb fusion proteins were purified by affinity chromatography using a glutathione-Sepharose 4B column, with final yields of 40 and 73 mg g(-1) dry cells, respectively. Specific activities were 15 and 20 mM ascorbate min(-1) mg(-1) protein, respectively. The K(m) values for ascorbate were 4 and 1 mM, respectively, and those for H(2)O(2) were 0.3 and 0.7 mM, respectively indicating that the two rice isoenzymes have different properties.
Lu
Zhenqiang
Z
Alkali Soil Natural Environmental Science Center (ASNESC), Stress Molecular Biology Laboratory, Northeast Forestry University, 150040, Harbin, P.R. China.
Takano
Tetsuo
T
Liu
Shenkui
S
D060387:Ascorbate Peroxidases; D001483:Base Sequence; D002846:Chromatography, Affinity; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology*; D010544:Peroxidases / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D005982:Glutathione Transferase