Expression and purification of Salmonella typhimurium RcsCDB system proteins.

MARIA DE LAS MERCEDES PESCARETTI; FABIAN E. LOPEZ; ROBERTO MORERO; MONICA A. DELGADO

Puerto Madryn

Congreso; XLVI Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology (SAIB).; 2010

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.