title: Expression and purification of SfaX(II), a protein involved in regulating adhesion and motility genes in extraintestinal pathogenic Escherichia coli.
abstract: Pathogenic Escherichia coli strains commonly harbor genes involved in formation of fimbriae, such as the sfa(II) fimbrial gene cluster found in uropathogenic and newborn meningitis isolates. The sfaX(II) gene, located at the distal end of the sfa(II) operon, was recently shown to play a role in controlling virulence-related gene expression in extraintestinal pathogenic E. coli (ExPEC). Until now, detailed characterization of the SfaX(II) protein has been hampered by difficulties in obtaining large quantities of soluble protein. By a rational modeling approach, we engineered a Cys70Ser mutation, which successfully improved solubility of the protein. Here, we present the expression, purification, and initial characterization of the recombinant SfaX(IIC70S) mutant. The protein was produced in E. coli BL21 (DE3) cells grown in autoinduction culture media. The plasmid vector harbored DNA encoding the SfaX(IIC70S) protein N-terminally fused with a six histidine (H6) sequence followed by a ZZ tag (a derivative of the Staphylococcus protein A) (H6-ZZ tag). The H6-ZZ tag was cleaved off with Tobacco Etch Virus (TEV) protease and the 166 amino acid full-length homo-dimeric protein was purified using affinity and size-exclusion chromatography. Electrophoretic mobility gel shift assays and atomic force microscopy demonstrated that the protein possesses DNA-binding properties, suggesting that the transcriptional regulatory activity of SfaX(II) can be mediated via direct binding to DNA.
authors.affiliation: Department of Molecular Biology, Umeå University, Umeå, Sweden.
authors.forename: Patricia
authors.initials: P
authors.lastname: Paracuellos
authors.forename: Anders
authors.initials: A
authors.lastname: Ohman
authors.forename: A Elisabeth
authors.initials: AE
authors.lastname: Sauer-Eriksson
authors.forename: Bernt Eric
authors.initials: BE
authors.lastname: Uhlin
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010861:Fimbriae, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D016415:Sequence Alignment
chemical_list: D029968:Escherichia coli Proteins