HIGH-LEVEL EXPRESSION AND PURIFICATION OF A HUMAN MINI-HEXOKINASE

Human hexokinase type I is a 100-kDa enzyme with the catalytic site lo cated in the C-terminal domain. We had previously expressed this domai n in Escherichia coli, however only a small amount of the recombinant enzyme was catalytically active. To overcome this problem we have now expressed the ''mini''-hexokinase using the pET expression system. An average of 1000 U of enzyme per liter of culture was obtained. The rec ombinant enzyme was purified to homogeneity by a combination of ion-ex change chromatography, affinity chromatography, and dye-ligand chromat ography. The enzyme was unstable under ultrafiltration; thus, a multic olumn purification procedure was developed in order to avoid the ultra filtration steps. The recombinant ''mini''-hexokinase was found to hav e the same kinetic properties as the entire enzyme. Using the method d escribed, the enzyme can be obtained in sufficient quantities for biop hysical and biochemical investigations. (C) 1996 Academic Press, Inc.