MAMMALIAN FERROCHELATASE - OVEREXPRESSION IN ESCHERICHIA-COLI AS A SOLUBLE-PROTEIN, PURIFICATION AND CHARACTERIZATION

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), a membrane bound p rotein, catalyzes the terminal step of the heme biosynthesis in all li ving systems. A cDNA encoding the murine ferrochelatase (Taketani, S., Nakahashi, Y., Osumi, T, and Tokunaga, R. (1990) J. Biol. Chem. 265, 19377-19380) has been expressed in Escherichia coli, using the alkalin e phosphatase promoter. Ferrochelatase was not only overexpressed in a n active form, but more importantly, was produced as a ''soluble prote in'' (i.e. associated with the soluble bacterial protein fraction). A simple purification from the ferrochelatase overproducing bacterial st rain yielded similar to 50 mg of protein/2-3 liters of bacterial cultu re. Recombinant ferrochelatase exhibited identical physical and cataly tic properties to those of mammalian ferrochelatases. Specifically, th e recombinant ferrochelatase has iron and porphyrin as substrates, and N-methylprotoporphyrin and metal ions (e.g. Hg2+ and Mn2+), as strong inhibitors of its enzyme activity. The K-m values are 112.5 mu m for iron and 95 mu m for deuteroporphyrin IX which are in the same range o f the K-m values determined for the ferrochelatases isolated from natu ral sources. This report describes the overexpression of a mammalian f errochelatase in E. coli, as a soluble protein, and its purification f rom an overproducing strain. The production of a functional and ''solu ble'' ferrochelatase has significance for the pursuit of structural an d functional studies of this enzyme.