Mammalian ferrochelatase. Overexpression in Escherichia coli as a soluble protein, purification and characterization.
Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), a membrane-bound protein, catalyzes the terminal step of the heme biosynthesis in all living 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 alkaline phosphatase promoter. Ferrochelatase was not only overexpressed in an active form, but more importantly, was produced as a "soluble protein" (i.e. associated with the soluble bacterial protein fraction). A simple purification from the ferrochelatase overproducing bacterial strain yielded approximately 50 mg of protein/2-3 liters of bacterial culture. Recombinant ferrochelatase exhibited identical physical and catalytic properties to those of mammalian ferrochelatases. Specifically, the 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 Km values are 112.5 microM for iron and 95 microM for deuteroporphyrin IX, which are in the same range of the Km values determined for the ferrochelatases isolated from natural sources. This report describes the overexpression of a mammalian ferrochelatase in E. coli, as a soluble protein, and its purification from an overproducing strain. The production of a functional and "soluble" ferrochelatase has significance for the pursuit of structural and functional studies of this enzyme.
Ferreira
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GC
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa 33612.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002413:Cations, Divalent; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli; D005294:Ferrochelatase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011524:Protoporphyrins / Q000494:pharmacology; D011994:Recombinant Proteins
D002413:Cations, Divalent; D017931:DNA Primers; D008670:Metals; D011524:Protoporphyrins; D011994:Recombinant Proteins; C028842:N-methylprotoporphyrin IX; D005294:Ferrochelatase