HIGH-LEVEL PRODUCTION AND PURIFICATION OF ESCHERICHIA-COLI N-ACETYLNEURAMINIC ACID ALDOLASE (EC-4.1.3.3)

TheEscherichia coli gene which encodesN-acetylneuraminic acid aldolase was isolated by the polymerase chain reaction, cloned into the inducible expression vector pTTQ18, and overexpressed inE. coli. The high yield of aldolase was achieved through both optimum growth of cells and efficient expression of the aldolase gene (20-30% soluble cellular protein). The recombinant enzyme was purified to homogeneity with an activity of 1.2-2.2 U/mg, which compared favorably with that of commercial preparations ofE. coli aldolase (1.1 U/mg) andClostridium perfringens aldolase (0.4 U/mg). The cloning strategy, fermentation conditions, purification protocol, and activity assay are described. © 1992 Academic Press, Inc.