HIGH-LEVEL EXPRESSION, PURIFICATION, AND CRYSTALLIZATION OF RECOMBINANT SPINACH GLYCOLATE OXIDASE IN ESCHERICHIA-COLI

Glycolate oxidase is a flavin-dependent enzyme in the photorespiratory pathway in plants. Here we report the heterologous expression of glyc olate oxidase in Escherichia coli and an isolation procedure which res ults in 4 mg pure protein per gram cell paste in only 1.5 days. This c orresponds to a more than 50-fold improvement in yield compared to pre viously reported expression systems. The purified recombinant protein can be crystallized easily, and the crystals are isomorphous to those obtained from the protein isolated from spinach, The availability of l arge amounts of enzyme will be a great advantage in the 3D structural and biochemical studies of mutants and inhibitor complexes. (C) 1996 A cademic Press, Inc.