Expression of human brain hexokinase in Escherichia coli: purification and characterization of the expressed enzyme

Biochem Biophys Res Commun. 1991 May 31;177(1):305-11. doi: 10.1016/0006-291x(91)91983-j.

Abstract

Human brain hexokinase (hexokinase I) was produced in Escherichia coli from a synthetic gene under control of the bacteriophage T7 promoter. The expressed coding region derives from a human cDNA clone thought to specify hexokinase I based on amino acid sequence identity between the predicted translation product and hexokinase I from rat brain. The open reading frame from this cDNA was fused to the promoter and 5' flanking region of T7 gene 10, and expressed in E. coli by induction of T7 RNA polymerase. Induced cells contained a hexokinase activity and an abundant protein of apparent molecular weight 100,000, neither of which was present in cells lacking T7 RNA polymerase. Enzyme purified to near homogeneity consisted of a 100,000 Da protein, the size predicted from the nucleotide sequence of the expressed cDNA. The purified enzyme had Michaelis constants of 32 microM and 0.3 mM for glucose and ATP, respectively, and bound to rat liver mitochondria in the presence of MgCl2. Enzymatic activity was inhibited by glucose-6-P and this inhibition was relieved by inorganic phosphate. Deinhibition by phosphate is a property specific to brain hexokinase.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Brain / enzymology*
  • Chromatography, High Pressure Liquid / methods
  • Chromatography, Ion Exchange / methods
  • Cloning, Molecular / methods
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics*
  • Genes, Synthetic
  • Hexokinase / genetics
  • Hexokinase / isolation & purification
  • Hexokinase / metabolism*
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • Mutagenesis, Site-Directed
  • Oligonucleotide Probes
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Restriction Mapping

Substances

  • Oligonucleotide Probes
  • Recombinant Proteins
  • Hexokinase