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. 1996 Mar 1;314(Pt 2):463–467. doi: 10.1042/bj3140463

Optimized heterologous expression of the human zinc enzyme glyoxalase I.

M Ridderström 1, B Mannervik 1
PMCID: PMC1217073  PMID: 8670058

Abstract

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.

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Selected References

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