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Id: lil-479676
Autor: Carvalho, H; Meneghini, R.
Título: Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL
Fonte: Braz. j. med. biol. res = Rev. bras. pesqui. méd. biol;41(4):270-276, Apr. 2008. ilus.
Idioma: en.
Resumo: 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.
Descritores: Chaperonina 10
Chaperonina 60
Escherichia coli/metabolismo
Proteína 1 Reguladora do Ferro/metabolismo
-Chaperonina 10
Chaperonina 60
Cromatografia por Troca Iônica
Ensaio de Desvio de Mobilidade Eletroforética
Escherichia coli/genética
Expressão Gênica
Proteína 1 Reguladora do Ferro/isolamento & purificação
Proteínas de Ligação a RNA
Solubilidade
Limites: Humanos
Tipo de Publ: Research Support, Non-U.S. Gov't
Responsável: BR1.1 - BIREME



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