Expression and purification of Salmonella typhimurium RcsCDB system proteins
- Autores
- Pescaretti, María de Las Mercedes; Farizano, Juan Vicente; Morero, Roberto Dionisio; Delgado, Monica Alejandra
- Año de publicación
- 2010
- Idioma
- inglés
- Tipo de recurso
- documento de conferencia
- Estado
- versión publicada
- Descripción
- The Rcs phosphorelay system involves the sensor protein RcsC, the cognate response regulator RcsB, and the histidin-containing phosphotransfer protein RcsD, which serve as an intermediary in the phosphoryl transfer from RcsC to RcsB. Previously, we reported that in the double mutant rcsD rcsC, the overproduction of RcsB regulator can not promote the Rcs system activation. These results suggested that only RcsB-P, the RcsB active form, is able to induce the RcsB-dependent genes modulation. We are interested to determinate if RcsC or RcsD can independently transfer the phosphate group to RcsB, or if in this process are necessary that both protein act together. In order to obtain soluble proteins, the full length rcsB gene and the sequences encoding the cytoplasmic domain of RcsC and RcsD, labeled with a His6 tag, were cloned into pT7-7 vector. The recombinant plasmids obtained were sequenced and the RcsB, RcsCcyt and RcsDcyt were expressed in E. coli BL21 DE3 strain. In the present work we performed the proteins purification step using Ni2+ affinity chromatography. The quality and quantity of the purified proteins were monitored by SDS-PAGE and BSA assay. The soluble proteins will be used an in vitro phosphorylation assays to determine the phosphorelay mechanism of the Rcs system.
Fil: Pescaretti, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Farizano, Juan Vicente. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Morero, Roberto Dionisio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular
Puerto Madryn
Argentina
Sociedad Argentina de Investigación Bioquímica y Biología Molecular - Materia
-
EXPRESSION
PURIFICATION
RCSCDB
PROTEINS - 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:11336/194945
Ver los metadatos del registro completo
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Expression and purification of Salmonella typhimurium RcsCDB system proteinsPescaretti, María de Las MercedesFarizano, Juan VicenteMorero, Roberto DionisioDelgado, Monica AlejandraEXPRESSIONPURIFICATIONRCSCDBPROTEINShttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The Rcs phosphorelay system involves the sensor protein RcsC, the cognate response regulator RcsB, and the histidin-containing phosphotransfer protein RcsD, which serve as an intermediary in the phosphoryl transfer from RcsC to RcsB. Previously, we reported that in the double mutant rcsD rcsC, the overproduction of RcsB regulator can not promote the Rcs system activation. These results suggested that only RcsB-P, the RcsB active form, is able to induce the RcsB-dependent genes modulation. We are interested to determinate if RcsC or RcsD can independently transfer the phosphate group to RcsB, or if in this process are necessary that both protein act together. In order to obtain soluble proteins, the full length rcsB gene and the sequences encoding the cytoplasmic domain of RcsC and RcsD, labeled with a His6 tag, were cloned into pT7-7 vector. The recombinant plasmids obtained were sequenced and the RcsB, RcsCcyt and RcsDcyt were expressed in E. coli BL21 DE3 strain. In the present work we performed the proteins purification step using Ni2+ affinity chromatography. The quality and quantity of the purified proteins were monitored by SDS-PAGE and BSA assay. The soluble proteins will be used an in vitro phosphorylation assays to determine the phosphorelay mechanism of the Rcs system.Fil: Pescaretti, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Farizano, Juan Vicente. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Morero, Roberto Dionisio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaXLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología MolecularPuerto MadrynArgentinaSociedad Argentina de Investigación Bioquímica y Biología MolecularSociedad Argentina de Investigación en Bioquímica y Biología Molecular2010info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectReuniónJournalhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/194945Expression and purification of Salmonella typhimurium RcsCDB system proteins; XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; Puerto Madryn; Argentina; 2010; 106-1060327-95451667-5746CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://saib.org.ar/archivos/biocell-34.pdfNacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-15T15:10:26Zoai:ri.conicet.gov.ar:11336/194945instacron: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 15:10:27.285CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
dc.title.none.fl_str_mv |
Expression and purification of Salmonella typhimurium RcsCDB system proteins |
title |
Expression and purification of Salmonella typhimurium RcsCDB system proteins |
spellingShingle |
Expression and purification of Salmonella typhimurium RcsCDB system proteins Pescaretti, María de Las Mercedes EXPRESSION PURIFICATION RCSCDB PROTEINS |
title_short |
Expression and purification of Salmonella typhimurium RcsCDB system proteins |
title_full |
Expression and purification of Salmonella typhimurium RcsCDB system proteins |
title_fullStr |
Expression and purification of Salmonella typhimurium RcsCDB system proteins |
title_full_unstemmed |
Expression and purification of Salmonella typhimurium RcsCDB system proteins |
title_sort |
Expression and purification of Salmonella typhimurium RcsCDB system proteins |
dc.creator.none.fl_str_mv |
Pescaretti, María de Las Mercedes Farizano, Juan Vicente Morero, Roberto Dionisio Delgado, Monica Alejandra |
author |
Pescaretti, María de Las Mercedes |
author_facet |
Pescaretti, María de Las Mercedes Farizano, Juan Vicente Morero, Roberto Dionisio Delgado, Monica Alejandra |
author_role |
author |
author2 |
Farizano, Juan Vicente Morero, Roberto Dionisio Delgado, Monica Alejandra |
author2_role |
author author author |
dc.subject.none.fl_str_mv |
EXPRESSION PURIFICATION RCSCDB PROTEINS |
topic |
EXPRESSION PURIFICATION RCSCDB PROTEINS |
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 Rcs phosphorelay system involves the sensor protein RcsC, the cognate response regulator RcsB, and the histidin-containing phosphotransfer protein RcsD, which serve as an intermediary in the phosphoryl transfer from RcsC to RcsB. Previously, we reported that in the double mutant rcsD rcsC, the overproduction of RcsB regulator can not promote the Rcs system activation. These results suggested that only RcsB-P, the RcsB active form, is able to induce the RcsB-dependent genes modulation. We are interested to determinate if RcsC or RcsD can independently transfer the phosphate group to RcsB, or if in this process are necessary that both protein act together. In order to obtain soluble proteins, the full length rcsB gene and the sequences encoding the cytoplasmic domain of RcsC and RcsD, labeled with a His6 tag, were cloned into pT7-7 vector. The recombinant plasmids obtained were sequenced and the RcsB, RcsCcyt and RcsDcyt were expressed in E. coli BL21 DE3 strain. In the present work we performed the proteins purification step using Ni2+ affinity chromatography. The quality and quantity of the purified proteins were monitored by SDS-PAGE and BSA assay. The soluble proteins will be used an in vitro phosphorylation assays to determine the phosphorelay mechanism of the Rcs system. Fil: Pescaretti, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Farizano, Juan Vicente. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Morero, Roberto Dionisio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular Puerto Madryn Argentina Sociedad Argentina de Investigación Bioquímica y Biología Molecular |
description |
The Rcs phosphorelay system involves the sensor protein RcsC, the cognate response regulator RcsB, and the histidin-containing phosphotransfer protein RcsD, which serve as an intermediary in the phosphoryl transfer from RcsC to RcsB. Previously, we reported that in the double mutant rcsD rcsC, the overproduction of RcsB regulator can not promote the Rcs system activation. These results suggested that only RcsB-P, the RcsB active form, is able to induce the RcsB-dependent genes modulation. We are interested to determinate if RcsC or RcsD can independently transfer the phosphate group to RcsB, or if in this process are necessary that both protein act together. In order to obtain soluble proteins, the full length rcsB gene and the sequences encoding the cytoplasmic domain of RcsC and RcsD, labeled with a His6 tag, were cloned into pT7-7 vector. The recombinant plasmids obtained were sequenced and the RcsB, RcsCcyt and RcsDcyt were expressed in E. coli BL21 DE3 strain. In the present work we performed the proteins purification step using Ni2+ affinity chromatography. The quality and quantity of the purified proteins were monitored by SDS-PAGE and BSA assay. The soluble proteins will be used an in vitro phosphorylation assays to determine the phosphorelay mechanism of the Rcs system. |
publishDate |
2010 |
dc.date.none.fl_str_mv |
2010 |
dc.type.none.fl_str_mv |
info:eu-repo/semantics/publishedVersion info:eu-repo/semantics/conferenceObject Reunión Journal http://purl.org/coar/resource_type/c_5794 info:ar-repo/semantics/documentoDeConferencia |
status_str |
publishedVersion |
format |
conferenceObject |
dc.identifier.none.fl_str_mv |
http://hdl.handle.net/11336/194945 Expression and purification of Salmonella typhimurium RcsCDB system proteins; XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; Puerto Madryn; Argentina; 2010; 106-106 0327-9545 1667-5746 CONICET Digital CONICET |
url |
http://hdl.handle.net/11336/194945 |
identifier_str_mv |
Expression and purification of Salmonella typhimurium RcsCDB system proteins; XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; Puerto Madryn; Argentina; 2010; 106-106 0327-9545 1667-5746 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://saib.org.ar/archivos/biocell-34.pdf |
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 application/pdf |
dc.coverage.none.fl_str_mv |
Nacional |
dc.publisher.none.fl_str_mv |
Sociedad Argentina de Investigación en Bioquímica y Biología Molecular |
publisher.none.fl_str_mv |
Sociedad Argentina de Investigación en Bioquímica y Biología Molecular |
dc.source.none.fl_str_mv |
reponame:CONICET Digital (CONICET) instname:Consejo Nacional de Investigaciones Científicas y Técnicas |
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CONICET Digital (CONICET) |
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Consejo Nacional de Investigaciones Científicas y Técnicas |
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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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