Data/hora: 11/06/2024 13:16:57

Provedor de dados: | 56
País: | Brazil
Título: | Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL
Autores: | Carvalho,H.; Meneghini,R.
Data: | 2008-04-01
Ano: | 2008
Palavras-chave: | Iron metabolism; Iron-regulatory protein 1; Chaperonins; GroES; GroEL; Protein purification.
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.
Tipo: | Info:eu-repo/semantics/article
Idioma: | Inglês
Identificador: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2008000400003
Editor: | Associação Brasileira de Divulgação Científica
Relação: | 10.1590/S0100-879X2008005000009
Formato: | text/html
Fonte: | Brazilian Journal of Medical and Biological Research  v.41 n.4 2008
Fonte: | info:eu-repo/semantics/openAccess