Expression, purification, and characterization of two NADP-malic enzymes of rice (Oryza sativa L.) in Escherichia coli.

Abstract NADP-malic enzymes (NADP-ME) are isozymes in plants. To clarify the diversity and function of NADP-ME isozymes in rice, we produced two active GST-fused NADP-ME proteins, NADP-ME 2 and NADP-ME 3 in Escherichia coli , and the fusion proteins were purified by affinity chromatography using a glutathione–Sepharose 4B column. After enzymatic cleavage of the GST tag, final yields were 1.4 mg/g wet cell weight (wcw) for NADP-ME 2 and 3.5 mg/g wcw for NADP-ME 3 , respectively, and the molecular weights of NADP-ME 2 and NADP-ME 3 were about 65 and 62 kDa, respectively. The optimum pH is 7.3 for NADP-ME 2 and 7.7 for NADP-ME 3 . The K m values for malate of NADP-ME 2 and NADP-ME 3 were 2.6 and 3.1 mM, whereas the K m values for NADP were 79 and 93 μM, respectively. The K cat values of NADP-ME 2 and NADP-ME 3 for malate were about 91.7 and 96.7 s −1 , respectively, and the K cat values for NADP about 88.3 and 98.3 s −1 , respectively. These results suggest that the two rice isozymes of NADP-ME in vitro have similar kinetic parameter.