Expression, purification and characterization of Oryza sativa L. NAD-malic enzyme in Escherichia coli

Abstract

The cDNA fragment of a rice NAD-malic enzyme (OsNAD-ME 1) was cloned and constructed into expression vector (pGEX-6p-3). OsNAD-ME 1 was successfully expressed as a GST fusion protein in Escherichia coli BL21. The optimal concentration of IPTG for inducement was 1 mmol/L and the optimal culture temperature was 30°C. The fusion protein was purified by using affinity chromatography with a glutathione sepharose 4B column. After enzymatic cleavage of GST tag, the OsNAD-ME 1 recombinant protein was collected for studying its kinetic properties. The optimum pH and temperature for catalytic reaction of OsNAD-ME 1 were pH 6.4 and 35°C, respectively. The k cat value determined at pH 6.4 was 36.38 s-1 and the K m values for NAD + and malate were 0.10 and 15.98 mmol/L, respectively. The maximum activity of OsNAD-ME 1 using NADP + as coenzyme was 64.47% of that using NAD + as coenzyme.

Roles of malic enzymes in plant development and stress responses

“…The NAD-ME activity in C3 plant rice increased with increasing temperature within a certain temperature range. 57 Under the condition of temperature difference in a certain temperature range, plants can adapt to the change of temperature through their own regulation. In the range of 22-55 ℃, the activities of NADP-ME increased with the increase of temperature.…”