One-step purification of a fully active hexahistidine-tagged human hexokinase type I overexpressed in Escherichia coli

The conversion of glucose into glucose 6-phosphate (Glc 6-P)(1) traps gluco se in a chemical state in which it cannot leave the cell and hence commits glucose to metabolism. In human tissues there are at least three hexokinase isoenzymes responsible for hexose phosphorylation. These enzymes are const ituted by a single polypeptide chain with a molecular weight of approximate ly 100 kDa, Among these isoenzymes, hexokinase type I is the most widely ex pressed in mammalian tissues and shows reversion of Glc 6-P inhibition by p hysiological levels of inorganic phosphate. In this work the hexokinase I f rom human brain was overexpressed in Escherichia coli, as a hexahistidine-t agged protein with the tag extending the C-terminal end. An average of 900 U per liter of culture was obtained. The expressed protein was one-step pur ified by metal chelate affinity chromatography performed in NTA-agarose col umn charged with Ni2+ ions. In order to stabilize the enzymatic activity 0. 5 RI ammonium sulfate was added to elution buffer. The specific activity of purified hexokinase I was 67.8 U/mg, The recombinant enzyme shows kinetic properties in agreement with those described for the native enzyme, and thu s it can be used for biophysical and biochemical investigation. (C) 2001 Ac ademic Press.