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
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/194945

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oai_identifier_str oai:ri.conicet.gov.ar:11336/194945
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repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling 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
reponame_str CONICET Digital (CONICET)
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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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