title: Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL.
abstract: Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1) plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.
authors.affiliation: Departamento de Ciências Biológicas, Universidade Federal de São Paulo, Diadema, SP, Brasil. helotonio.carvalho@gmail.com
authors.forename: H
authors.initials: H
authors.lastname: Carvalho
authors.forename: R
authors.initials: R
authors.lastname: Meneghini
mesh_terms: D018835:Chaperonin 10 / Q000235:genetics / Q000378:metabolism*; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D035941:Iron Regulatory Protein 1 / Q000302:isolation & purification / Q000378:metabolism*; D016601:RNA-Binding Proteins; D012995:Solubility
chemical_list: D018835:Chaperonin 10; D018834:Chaperonin 60; D016601:RNA-Binding Proteins; D035941:Iron Regulatory Protein 1