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-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 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-ME₁ recombinant protein was collected for studying its kinetic properties. The optimum pH and temperature for catalytic reaction of OsNAD-ME₁ 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-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.