Optimized heterologous expression of the human zinc enzyme glyoxalase I.
DNA coding for human glyoxalase I was isolated from a HeLa cell cDNA library by means of PCR. The deduced amino acid sequence differs form previously isolated sequences in that a glutamic acid replaces an alanine in position 111. This variant cDNA may represent the more acidic isoform of glyoxalase I originally identified at the protein level. An expression clone was constructed for high-level production of glyoxalase I in Escherichia coli. For optimal yield of the recombinant protein, silent random mutations were introduced in the cDNA coding region. Antisera against human glyoxalase I were used to select a high-level expression clone. This clone afforded 60 mg of purified enzyme per litre of culture medium. Addition of a zinc salt to the culture medium was essential to obtain an active enzyme and a stoicheiometric metal content. The functional characterization of the recombinant enzyme included determination of kinetic constants for methylglyoxal, phenylglyoxal and p-phenylphenylglyoxal, as well as inhibition studies. The kinetic properties of recombinant glyoxalase I were indistinguishable from those of the enzyme purified from human tissues.
Ridderström
M
M
Department of Biochemistry, Uppsala University, Biomedical Center, Sweden.
Mannervik
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D007791:Lactoylglutathione Lyase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D007791:Lactoylglutathione Lyase; D015032:Zinc