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

摘要

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, and NADP-ME3 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-ME2 and 3.5 mg/g wcw for NADP-ME3, respectively, and the molecular weights of NADP-ME2 and NADP-ME3 were about 65 and 62 kDa, respectively. The optimum pH is 7.3 for NADP-ME2 and 7.7 for NADP-ME3. The K-m values for malate of NADP-ME2 and NADP-ME3 were 2.6 and 3.1 mM, whereas the K-m values for NADP were 79 and 93 mu M, respectively. The K-cat values of NADP-ME2 and NADP-ME3 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. (C) 2005 Elsevier Inc. All rights reserved.