HETEROLOGOUS EXPRESSION AND CHARACTERIZATION OF SOYBEAN CYTOSOLIC ASCORBATE PEROXIDASE

Ascorbate peroxidase is a widespread plant enzyme that catalyzes the r emoval of potentially harmful H2O2. This enzyme is particularly import ant in legume root nodules due to their high potential for generating activated forms of oxygen, A cDNA clone of soybean nodule ascorbate pe roxidase was used to construct an expression system in Escherichia coi l. The recombinant protein had an N-terminal tag of six consecutive hi stidine residues to allow for purification by Ni2+-agarose affinity ch romatography. Large amounts of recombinant peroxidase (about 27% of to tal soluble protein) were produced but most of the peroxidase was pres ent in the ape-form (without heme). Addition of delta-aminolevulinic a cid to the growth media resulted in an increase in production of holop rotein, Apoprotein was easily converted to the hole-form by in vitro r econstitution with hemin. The reconstituted protein was catalytically, spectrally, and immunologically indistinguishable from native ascorba te peroxidase. (C) 1996 Academic Press, Inc.