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
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/48131
Ver los metadatos del registro completo
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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 |
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1846082830652669952 |
score |
13.22299 |