High-level expression and purification of a human "mini"-hexokinase.
Human hexokinase type I is a 100-kDa enzyme with the catalytic site located in the C-terminal domain. We had previously expressed this domain 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 recombinant enzyme was purified to homogeneity by a combination of ion-exchange chromatography, affinity chromatography, and dye-ligand chromatography. The enzyme was unstable under ultrafiltration; thus, a multicolumn purification procedure was developed in order to avoid the ultrafiltration steps. The recombinant "mini"-hexokinase was found to have the same kinetic properties as the entire enzyme. Using the method described, the enzyme can be obtained in sufficient quantities for biophysical and biochemical investigations.
Bianchi
M
M
Insititute of Biological Chemistry, University of Urbino, Italy.
Serafini
G
G
Corsi
D
D
Magnani
M
M
D015153:Blotting, Western; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006593:Hexokinase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion
D011994:Recombinant Proteins; D006593:Hexokinase