MOLECULAR-CLONING, HETEROLOGOUS EXPRESSION, AND CHARACTERIZATION OF HUMAN GLYOXALASE-II

A clone encoding glyoxalase II has been isolated from a human adult li ver cDNA library, The sequence of 1011 base pairs consists of a full-l ength coding region of 780 base pairs, corresponding to a protein with a calculated molecular mass of 28,861 daltons. Identities (50-60%) we re found to partial 5' and 3' cDNA sequences from Arabidopsis thaliana as well as within a limited region of glutathione transferase I cDNA from corn, A vector was constructed for heterologous expression of gly oxalase II in Escherichia coli. For optimal yield of enzyme, silent ra ndom mutations were introduced in the 5' coding region of the cDNA. A yield of 25 mg of glyoxalase II per liter of culture medium was obtain ed after affinity purification with immobilized glutathione. The recom binant enzyme had full catalytic activity and kinetic parameters indis tinguishable from those of the native enzyme purified from human eryth rocytes.