Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL
Autor(a) principal: | |
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Data de Publicação: | 2008 |
Outros Autores: | |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNIFESP |
Texto Completo: | http://repositorio.unifesp.br/handle/11600/4321 http://dx.doi.org/10.1590/S0100-879X2008005000009 |
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. |
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Carvalho, Helotonio [UNIFESP]Meneghini, RogérioUniversidade Federal de São Paulo (UNIFESP)Centro Latino-Americano e do Caribe de Informações em Ciências da Saúde2015-06-14T13:38:29Z2015-06-14T13:38:29Z2008-04-01Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 41, n. 4, p. 270-276, 2008.0100-879Xhttp://repositorio.unifesp.br/handle/11600/4321http://dx.doi.org/10.1590/S0100-879X2008005000009S0100-879X2008000400003.pdfS0100-879X200800040000310.1590/S0100-879X2008005000009WOS:000254737100003Iron 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.Universidade Federal de São Paulo (UNIFESP) Departamento de Ciências BiológicasCentro Latino-Americano e do Caribe de Informações em Ciências da SaúdeUNIFESP, Depto. de Ciências BiológicasSciELO270-276engAssociação Brasileira de Divulgação CientíficaBrazilian Journal of Medical and Biological ResearchIron metabolismIron-regulatory protein 1ChaperoninsGroESGroELProtein purificationIncreased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroELinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESPORIGINALS0100-879X2008000400003.pdfapplication/pdf785543${dspace.ui.url}/bitstream/11600/4321/1/S0100-879X2008000400003.pdf68d27d63eedc46d4730d001e93008a1bMD51open accessTEXTS0100-879X2008000400003.pdf.txtS0100-879X2008000400003.pdf.txtExtracted texttext/plain27867${dspace.ui.url}/bitstream/11600/4321/9/S0100-879X2008000400003.pdf.txt52c63ff4687702c0b5317007178896caMD59open accessTHUMBNAILS0100-879X2008000400003.pdf.jpgS0100-879X2008000400003.pdf.jpgIM Thumbnailimage/jpeg6667${dspace.ui.url}/bitstream/11600/4321/11/S0100-879X2008000400003.pdf.jpgdd90fb1641d4b148191c0e5c08d5d0eaMD511open access11600/43212023-06-05 19:30:04.063open accessoai:repositorio.unifesp.br:11600/4321Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestopendoar:34652023-06-05T22:30:04Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false |
dc.title.en.fl_str_mv |
Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL |
title |
Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL |
spellingShingle |
Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL Carvalho, Helotonio [UNIFESP] Iron metabolism Iron-regulatory protein 1 Chaperonins GroES GroEL Protein purification |
title_short |
Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL |
title_full |
Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL |
title_fullStr |
Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL |
title_full_unstemmed |
Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL |
title_sort |
Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL |
author |
Carvalho, Helotonio [UNIFESP] |
author_facet |
Carvalho, Helotonio [UNIFESP] Meneghini, Rogério |
author_role |
author |
author2 |
Meneghini, Rogério |
author2_role |
author |
dc.contributor.institution.none.fl_str_mv |
Universidade Federal de São Paulo (UNIFESP) Centro Latino-Americano e do Caribe de Informações em Ciências da Saúde |
dc.contributor.author.fl_str_mv |
Carvalho, Helotonio [UNIFESP] Meneghini, Rogério |
dc.subject.eng.fl_str_mv |
Iron metabolism Iron-regulatory protein 1 Chaperonins GroES GroEL Protein purification |
topic |
Iron metabolism Iron-regulatory protein 1 Chaperonins GroES GroEL Protein purification |
description |
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. |
publishDate |
2008 |
dc.date.issued.fl_str_mv |
2008-04-01 |
dc.date.accessioned.fl_str_mv |
2015-06-14T13:38:29Z |
dc.date.available.fl_str_mv |
2015-06-14T13:38:29Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 41, n. 4, p. 270-276, 2008. |
dc.identifier.uri.fl_str_mv |
http://repositorio.unifesp.br/handle/11600/4321 http://dx.doi.org/10.1590/S0100-879X2008005000009 |
dc.identifier.issn.none.fl_str_mv |
0100-879X |
dc.identifier.file.none.fl_str_mv |
S0100-879X2008000400003.pdf |
dc.identifier.scielo.none.fl_str_mv |
S0100-879X2008000400003 |
dc.identifier.doi.none.fl_str_mv |
10.1590/S0100-879X2008005000009 |
dc.identifier.wos.none.fl_str_mv |
WOS:000254737100003 |
identifier_str_mv |
Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 41, n. 4, p. 270-276, 2008. 0100-879X S0100-879X2008000400003.pdf S0100-879X2008000400003 10.1590/S0100-879X2008005000009 WOS:000254737100003 |
url |
http://repositorio.unifesp.br/handle/11600/4321 http://dx.doi.org/10.1590/S0100-879X2008005000009 |
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eng |
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eng |
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Brazilian Journal of Medical and Biological Research |
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info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
270-276 |
dc.publisher.none.fl_str_mv |
Associação Brasileira de Divulgação Científica |
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Associação Brasileira de Divulgação Científica |
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