Purification of MBP-EnvZ fusion proteins using an automated system.
Bacteria use two-component signal transduction systems to detect and respond to environmental changes. These systems have been studied systematically in Escherichia coli as a model organism. Most of the signal transduction systems present in E. coli are conserved in related pathogenic bacteria; however, differences in regulation by these systems have been reported from one bacterial species to another [Oropeza, R., and Calva, E. (2009). The cysteine 354 and 277 residues of Salmonella enterica serovar Typhi EnvZ are determinants of autophosphorylation and OmpR phosphorylation. FEMS Microbiol. Lett.292, 282-290]. Our laboratory has been interested in studying the OmpR/EnvZ two-component system in S. enterica. In S. enterica serovar Typhi (Typhi), it regulates the expression of the porin genes, namely ompC, ompF, ompS1, and ompS2. OmpR proteins are identical between E. coli and Typhi, but several differences exist between the EnvZ proteins. To define whether some differences in porin regulation are due to changes on EnvZ, we decided to overexpress and purify E. coli, Typhi, and S. enterica serovar Typhimurium (Typhimurium) EnvZ proteins fused to the maltose-binding protein (MBP) as a purification tag. Differences in the autophosphorylation level of these proteins were evidenced. Hence, considering the differences at the amino acid level between E. coli and Typhi EnvZ proteins, several mutations were introduced in the Typhi EnvZ protein in order to try to find the amino acids affecting the enzymatic activity of the protein. We found that Cys354 plays an important role in defining the enzymatic activity of this histidine kinase. Here, we report the automated purification of a collection of MBP-EnvZ fusions using a mini-chromatography commercial system, but adapting an amylose affinity column packed by ourselves.
Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México AP 510-3, Cuernavaca, Morelos, Mexico.
Ricardo
R
Oropeza
Edmundo
E
Calva
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012485:Salmonella typhi / Q000235:genetics / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins