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 H2O2 were 0.3 and 0.7 mM, respectively indicating that the two rice isoenzymes have different properties.