Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex

Autores
Juri Ayub, Maximiliano; Levin, Mariano Jorge; Aguilar, Carlos Fernando
Año de publicación
2001
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The P0 protein is part of the ribosomal eukaryotic stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. P0 is the minimal portion of the stalk that is able to support accurate protein synthesis. The P0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by the Trypanosoma cruzi parasite. The T. cruzi P0 (TcP0) protein was cloned into the pRSET A vector and expressed in Escherichia coli fused to a His-tag. The identity of the protein was confirmed by immunoblotting. Due to the formation of inclusion bodies the protein was purified using the following steps: (i) differential centrifugation to separate the inclusion bodies from soluble proteins and (ii) affinity chromatography under denaturing conditions. TcP0 showed high tendency to aggregation during refolding assays. However, TcP0 could be efficiently folded in the presence of a low concentration of SDS. The folding of the protein was confirmed using urea gradient electrophoresis, limited proteolysis, circular dichroism, and tryptophan fluorescence. Native electrophoresis showed that the folded TcP0 (and not a folding intermediate) was the cause of aggregation in the absence of SDS. The protocol described here permitted us to obtain large amounts (up to 30 mg per culture liter) of pure and folded TcP0, a very hydrophobic protein with a high tendency to aggregation.
Fil: Juri Ayub, Maximiliano. Universidad Nacional de San Luis; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Levin, Mariano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Aguilar, Carlos Fernando. Universidad Nacional de San Luis; Argentina
Materia
Ribosomal P0 Protein
Trypanosoma Cruzi
P1/P2/P0 Complex
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/48131
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/48131
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complexJuri Ayub, MaximilianoLevin, Mariano JorgeAguilar, Carlos FernandoRibosomal P0 ProteinTrypanosoma CruziP1/P2/P0 Complexhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The P0 protein is part of the ribosomal eukaryotic stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. P0 is the minimal portion of the stalk that is able to support accurate protein synthesis. The P0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by the Trypanosoma cruzi parasite. The T. cruzi P0 (TcP0) protein was cloned into the pRSET A vector and expressed in Escherichia coli fused to a His-tag. The identity of the protein was confirmed by immunoblotting. Due to the formation of inclusion bodies the protein was purified using the following steps: (i) differential centrifugation to separate the inclusion bodies from soluble proteins and (ii) affinity chromatography under denaturing conditions. TcP0 showed high tendency to aggregation during refolding assays. However, TcP0 could be efficiently folded in the presence of a low concentration of SDS. The folding of the protein was confirmed using urea gradient electrophoresis, limited proteolysis, circular dichroism, and tryptophan fluorescence. Native electrophoresis showed that the folded TcP0 (and not a folding intermediate) was the cause of aggregation in the absence of SDS. The protocol described here permitted us to obtain large amounts (up to 30 mg per culture liter) of pure and folded TcP0, a very hydrophobic protein with a high tendency to aggregation.Fil: Juri Ayub, Maximiliano. Universidad Nacional de San Luis; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Levin, Mariano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Aguilar, Carlos Fernando. Universidad Nacional de San Luis; ArgentinaAcademic Press Inc Elsevier Science2001-06info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/48131Juri Ayub, Maximiliano; Levin, Mariano Jorge; Aguilar, Carlos Fernando; Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex; Academic Press Inc Elsevier Science; Protein Expression and Purification; 22; 2; 6-2001; 225-2331046-59281096-0279CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S1046592801914275info:eu-repo/semantics/altIdentifier/doi/10.1006/prep.2001.1427info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-15T14:36:29Zoai:ri.conicet.gov.ar:11336/48131instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-15 14:36:30.048CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex
title Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex
spellingShingle Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex
Juri Ayub, Maximiliano
Ribosomal P0 Protein
Trypanosoma Cruzi
P1/P2/P0 Complex
title_short Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex
title_full Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex
title_fullStr Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex
title_full_unstemmed Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex
title_sort Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex
dc.creator.none.fl_str_mv Juri Ayub, Maximiliano
Levin, Mariano Jorge
Aguilar, Carlos Fernando
author Juri Ayub, Maximiliano
author_facet Juri Ayub, Maximiliano
Levin, Mariano Jorge
Aguilar, Carlos Fernando
author_role author
author2 Levin, Mariano Jorge
Aguilar, Carlos Fernando
author2_role author
author
dc.subject.none.fl_str_mv Ribosomal P0 Protein
Trypanosoma Cruzi
P1/P2/P0 Complex
topic Ribosomal P0 Protein
Trypanosoma Cruzi
P1/P2/P0 Complex
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv The P0 protein is part of the ribosomal eukaryotic stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. P0 is the minimal portion of the stalk that is able to support accurate protein synthesis. The P0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by the Trypanosoma cruzi parasite. The T. cruzi P0 (TcP0) protein was cloned into the pRSET A vector and expressed in Escherichia coli fused to a His-tag. The identity of the protein was confirmed by immunoblotting. Due to the formation of inclusion bodies the protein was purified using the following steps: (i) differential centrifugation to separate the inclusion bodies from soluble proteins and (ii) affinity chromatography under denaturing conditions. TcP0 showed high tendency to aggregation during refolding assays. However, TcP0 could be efficiently folded in the presence of a low concentration of SDS. The folding of the protein was confirmed using urea gradient electrophoresis, limited proteolysis, circular dichroism, and tryptophan fluorescence. Native electrophoresis showed that the folded TcP0 (and not a folding intermediate) was the cause of aggregation in the absence of SDS. The protocol described here permitted us to obtain large amounts (up to 30 mg per culture liter) of pure and folded TcP0, a very hydrophobic protein with a high tendency to aggregation.
Fil: Juri Ayub, Maximiliano. Universidad Nacional de San Luis; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina
Fil: Levin, Mariano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Aguilar, Carlos Fernando. Universidad Nacional de San Luis; Argentina
description The P0 protein is part of the ribosomal eukaryotic stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. P0 is the minimal portion of the stalk that is able to support accurate protein synthesis. The P0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by the Trypanosoma cruzi parasite. The T. cruzi P0 (TcP0) protein was cloned into the pRSET A vector and expressed in Escherichia coli fused to a His-tag. The identity of the protein was confirmed by immunoblotting. Due to the formation of inclusion bodies the protein was purified using the following steps: (i) differential centrifugation to separate the inclusion bodies from soluble proteins and (ii) affinity chromatography under denaturing conditions. TcP0 showed high tendency to aggregation during refolding assays. However, TcP0 could be efficiently folded in the presence of a low concentration of SDS. The folding of the protein was confirmed using urea gradient electrophoresis, limited proteolysis, circular dichroism, and tryptophan fluorescence. Native electrophoresis showed that the folded TcP0 (and not a folding intermediate) was the cause of aggregation in the absence of SDS. The protocol described here permitted us to obtain large amounts (up to 30 mg per culture liter) of pure and folded TcP0, a very hydrophobic protein with a high tendency to aggregation.
publishDate 2001
dc.date.none.fl_str_mv 2001-06
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/48131
Juri Ayub, Maximiliano; Levin, Mariano Jorge; Aguilar, Carlos Fernando; Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex; Academic Press Inc Elsevier Science; Protein Expression and Purification; 22; 2; 6-2001; 225-233
1046-5928
1096-0279
CONICET Digital
CONICET
url http://hdl.handle.net/11336/48131
identifier_str_mv Juri Ayub, Maximiliano; Levin, Mariano Jorge; Aguilar, Carlos Fernando; Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex; Academic Press Inc Elsevier Science; Protein Expression and Purification; 22; 2; 6-2001; 225-233
1046-5928
1096-0279
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S1046592801914275
info:eu-repo/semantics/altIdentifier/doi/10.1006/prep.2001.1427
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Academic Press Inc Elsevier Science
publisher.none.fl_str_mv Academic Press Inc Elsevier Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1846082830652669952
score 13.22299

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