Expression, purification, and characterization of recombinant mammalian ferrochelatase

Abstract

Publisher Summary This chapter presents the expression, purification, and characterization of recombinant mammalian ferrochelatase. Human ferrochelatase is encoded by a single nuclear gene located at chromosome 18q21.3. Recombinant mammalian ferrochelatase can be assayed at any point from crude cell extract to purified enzyme. The standard assay used in the laboratory employs iron and porphyrin as substrates and quantitates the product as the pyridine hemochromogen. The physiological substrate, protoporphyrin IX, is used most frequently, yet mesoporphyrin, deuteroporphyrin, and hematoporphyrin are all suitable substrates. Plasmids encoding mouse or human ferrochelatase, minus the aminoterminal mitochondrial targeting sequence, are engineered with an additional upstream ribosomal binding site. Expression in Escherichia coli strain JM109 allows maximal protein production as compared to other strains tested. Escherichia coli strains JM109 and DH5 both have been found to be acceptable for production of holoenzyme. A variety of other available strains, including those that employ the inducible T7 promoter system, have been tested and found to be less acceptable. Some strains produce large amounts of apoprotein, as evidenced by sodium dodecyl sulfate (SDS) gel electrophoresis, but produce little active enzyme. Such strains, which do not contain the metal center, should be acceptable for expression of ferrochelatases.