A recombinant vector was constructed to overproduce a glutathione S-transferase (GST) tagged N-acetyl- d -neuraminic acid aldolase (Neu5Ac aldolase) fusion protein in Escherichia coli. A solid-phase cloning method was proposed for the recombination of a GST-containing plasmid and Neu5Ac aldolase gene amplified from E. coli K12. Although the over-expressed fusion protein was largely present as aggregate or inclusion body, soluble GST-Neu5Ac aldolase fusion protein possessing the enzymatic activity could be purified to homogeneity by the affinity of GST tag. Results from sequencing the reconstruction of Neu5Ac aldolase gene fusion and gel electrophoresis of its expressing product suggested that the insertion of DNA was in the proper reading frame. Using N-acetyl- d -neuraminic acid (Neu5Ac) as the substrate, the purified fusion protein showed a Km value of 2.8 mM that is close to those reported in the literature for Neu5Ac aldolase, indicating that the addition of GST tag in the N terminus did not change the affinity of Neu5Ac aldolase for substrate. The GST-Neu5Ac aldolase fusion protein, which was applied even in a very small dosage, could effectively catalyze the production of 2-keto-3-deoxy- d -glycero- d -galacto-nonopyranulosonic acid (KDN) from d -mannose and pyruvic acid.
