Expression of human brain hexokinase in Escherichiacoli: Purification and characterization of the expressed enzyme

Feng Liu, Lily Q Dong, Alan M. Myers, Herbert J. Fromm

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Original languageEnglish
Pages (from-to)305-311
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume177
Issue number1
DOIs
StatePublished - May 31 1991
Externally publishedYes

Fingerprint

Hexokinase
Purification
Brain
Enzymes
Complementary DNA
Rats
Genes
Phosphates
Synthetic Genes
Bacteriophage T7
Glucose
Magnesium Chloride
Mitochondria
Bacteriophages
Liver Mitochondrion
5' Flanking Region
Liver
Escherichia coli
Open Reading Frames
Amino Acid Sequence

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Expression of human brain hexokinase in Escherichiacoli : Purification and characterization of the expressed enzyme. / Liu, Feng; Dong, Lily Q; Myers, Alan M.; Fromm, Herbert J.

In: Biochemical and Biophysical Research Communications, Vol. 177, No. 1, 31.05.1991, p. 305-311.

Research output: Contribution to journalArticle

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abstract = "Human brain hexokinase (hexokinase I) was produced in Escherichiacoli 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 aboundant protein of apparent molecular weight 100,000, neither of which was present in cells lacking T7 RNA polymerase. Enzyme purified to nerr 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 μM 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.",
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T2 - Purification and characterization of the expressed enzyme

AU - Liu, Feng

AU - Dong, Lily Q

AU - Myers, Alan M.

AU - Fromm, Herbert J.

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N2 - Human brain hexokinase (hexokinase I) was produced in Escherichiacoli 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 aboundant protein of apparent molecular weight 100,000, neither of which was present in cells lacking T7 RNA polymerase. Enzyme purified to nerr 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 μM 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.

AB - Human brain hexokinase (hexokinase I) was produced in Escherichiacoli 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 aboundant protein of apparent molecular weight 100,000, neither of which was present in cells lacking T7 RNA polymerase. Enzyme purified to nerr 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 μM 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.

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