Titlebook: Iron Metabolism; Methods and Protocol Oleh Khalimonchuk Book 2024 The Editor(s) (if applicable) and The Author(s), under exclusive license

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Recombinant Biogenesis and Analysis of Cytochrome , Speciesesenting an avenue for the study of cytochrome . or the cytochrome . biogenesis pathways responsible for heme attachment. Here, we describe a method for recombinant . production of holocytochrome . utilizing the System I (CcmABCDEFGH) bacterial cytochrome . biogenesis pathway, followed by analysis o

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Ultra-Performance Liquid Chromatography (UPLC) Analysis of Heme Biosynthesis IntermediatesThe first product, ALA, was derivatized to a highly fluorescent pyrrolizine; PBG, the second intermediate, was enzymatically converted to uroporphyrinogen, and all the porphyrinogen intermediates were oxidized in acid to form fluorescent porphyrins. Heme was measured as hemin. The stable porphyrin?f

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Heterologous Expression and Purification of Eukaryotic ALA Synthase from al hexahistidine affinity tag followed by a SUMO fusion tag for solubility and ease of purification. The plasmid is introduced into competent . cells, and robust protein expression is induced with IPTG. The ALAS cofactor, pyridoxal 5′-phosphate, is inserted during protein production to yield an acti

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