EXPRESSION AND PURIFICATION OF Salmonella

PESCARETTI MM,; JUAN V. FARIZANO; MORERO RD,; DELGADO, MONICA ALEJANDRA

Puerto Madryn, Argentina

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigación en; 2010

Sociedad Argentina de Investigación en

EXPRESSION AND PURIFICATION OF Salmonella typhimurium RcsCDB SYSTEM PROTEINS typhimurium RcsCDB SYSTEM PROTEINS typhimurium RcsCDB SYSTEM PROTEINS typhimurium RcsCDB SYSTEM PROTEINS typhimurium RcsCDB SYSTEM PROTEINS typhimurium RcsCDB SYSTEM PROTEINS Salmonella typhimurium RcsCDB SYSTEM PROTEINSRcsCDB SYSTEM PROTEINS Pescaretti MM, Farizano JV, Morero RD, Delgado MA Dpto. Bqca. de la Nutrición-INSIBIO - Inst. Qca. Biológica (CONICET-UNT) Tucumán. E-mail: monaledel@hotmail.com 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 determine if RcsC or RcsD can independently transfer the phosphate group to RcsB, or if in this process is necessary that both proteins 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 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 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 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 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 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 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 determine if RcsC or RcsD can independently transfer the phosphate group to RcsB, or if in this process is necessary that both proteins 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 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 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 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 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 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 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 determine if RcsC or RcsD can independently transfer the phosphate group to RcsB, or if in this process is necessary that both proteins 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 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 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 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 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 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 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 determine if RcsC or RcsD can independently transfer the phosphate group to RcsB, or if in this process is necessary that both proteins 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 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 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 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 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 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 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 determine if RcsC or RcsD can independently transfer the phosphate group to RcsB, or if in this process is necessary that both proteins 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 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 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 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 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 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 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 determine if RcsC or RcsD can independently transfer the phosphate group to RcsB, or if in this process is necessary that both proteins 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 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 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 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 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 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 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 determine if RcsC or RcsD can independently transfer the phosphate group to RcsB, or if in this process is necessary that both proteins 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 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 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 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 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 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 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 2+ 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.