NAD+-dependent malic enzyme (NAD-ME) gene from Escherichia coli K12 was inserted into an expression vector pET24b(+) and transformed into E coli BL21 (DE3). Recombinant NAD-ME was expressed upon IPTG induction, purified with affinity chromatography, and biochemically characterized. The results showed that recombinant NAD-ME could be produced mainly in a soluble form. The monomeric molecular weight of recombinant NAD-ME was about 65 kDa, whereas monomer, homotetramer, and homooctamer were formed in solution as revealed by nondenaturing polyacrylamide gel electrophoresis analysis. Finally, the K-m, values of NAD-ME for L-malate and NAD were determined as 0.420 +/- 0.174 and 0.097 +/- 0.038 mM, respectively, at pH 7.2. By using this over-expression and purification system, recombinant E coli K12 NAD-ME can now be obtained in large quantity necessary for further biochemical characterization and applications. (c) 2006 Elsevier Inc. All rights reserved.