Expression, purification, and characterization of two NADP-malic enzymes of rice (Oryza sativa L.) in Escherichia coli.
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-ME2 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 Km values for malate of NADP-ME2 and NADP-ME3 were 2.6 and 3.1 mM, whereas the Km values for NADP were 79 and 93 microM, respectively. The Kcat values of NADP-ME2 and NADP-ME3 for malate were about 91.7 and 96.7 s-1, respectively, and the Kcat 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.
Cheng
Yuxiang
Y
Alkali Soil Natural Environmental Science Center (ASNESC), Stress Molecular Biology Laboratory, Northeast Forestry University, Harbin 150040, PR China.
Takano
Tetsuo
T
Zhang
Xinxin
X
Yu
Song
S
Liu
Dali
D
Liu
Shenkui
S
D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013997:Time Factors
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)