Overexpression in Escherichia coli, and one-step purification of the human recombinant ferrochelatase.
Ferrochelatase (EC 4.99.1.1), a mitochondrial inner membrane-bound protein, is the terminal enzyme of heme biosynthesis. The cDNA encoding the human mature ferrochelatase was placed under transcriptional control of T7 RNA polymerase in an Escherichia coli expression system. The bacteria produced large amounts of 42 kDa protein which reacted with anti-ferrochelatase antibodies. Expressed ferrochelatase exhibited iron- and zinc-chelating activities, and was found as a soluble protein. The recombinant enzyme has been purified to apparent homogeneity with a high yield, by one-step purification involving Blue-Sepharose chromatography. The purified enzyme which showed a molecular weight of about 40,000 by gel-filtration, functioned in a monomeric form. Km value for both mesoporphyrin IX and protoporphyrin IX with zinc was 12.5 microM. Km values for iron and zinc with mesoporphyrin IX were 6.7 microM and 11.8 microM, respectively. Zinc-chelating activity was markedly stimulated by palmitic acid, but iron-chelating activity remained unchanged. The above results were similar to those reported previously for mammalian ferrochelatase. The overexpression and the simple purification of a functional ferrochelatase exhibiting the same properties as natural enzyme will allow us to elucidate the mechanism of the enzyme reaction and structural changes of the mutated enzyme.
Okuda
M
M
Department of Hygiene, Kansai Medical University, Osaka, Japan.
Kohno
H
H
Furukawa
T
T
Tokunaga
R
R
Taketani
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005294:Ferrochelatase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification
D011994:Recombinant Proteins; D005294:Ferrochelatase