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

The cDNA fragment of a rice NAD-malic enzyme (OsNAD-ME1) was cloned and constructed into expression vector (pGEX-6p-3). OsNAD-ME1 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 culturetemperature 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-ME1 recombinant protein was collected for studying its kinetic properties. The optimum pH and temperature for catalytic reaction ofOsNAD-ME1 were pH 6.4 and 35°C, respectively. The kcat value determined at pH 6.4 was 36.38 s-1 and the Km values for NAD+ and malate were 0.10 and 15.98mmol/L, respectively. The maximum activity of OsNAD-ME1 using NADP+ as coenzyme was 64.47% of that using NAD+ as coenzyme.   Key words: Enzyme activity, GST fusion protein, kinetic properties, NAD-malic enzyme, Oryza sativa L., purification.