OPTIMIZED HETEROLOGOUS EXPRESSION OF THE HUMAN ZINC ENZYME GLYOXALASE-I

DNA coding for human glyoxalase I was isolated from a HeLa cell cDNA l ibrary by means of PCR. The deduced amino acid sequence differs from p reviously isolated sequences in that a glutamic acid replaces an alani ne in position 111. This variant cDNA may represent the more acidic is oform of glyoxalase I originally identified at the protein level. An e xpression clone was constructed for high-level production of glyoxalas e I in Escherichia coli. For optimal yield of the recombinant protein, silent random mutations were introduced in the cDNA coding region. An tisera against human glyoxalase I were used to select a high-level exp ression clone. This clone afforded 60 mg of purified enzyme per litre of culture medium. Addition of a zinc salt to the culture medium was e ssential to obtain an active enzyme and a stoicheiometric metal conten t. characterization of the recombinant enzyme mination of kinetic cons tants for methylglyoxal, phenylglyoxal and p-phenylphenylglyoxal, as w ell as inhibition studies. The kinetic properties of recombinant glyox alase I were indistinguishable from those of the enzyme purified from human tissues.