Expression and purification of SfaXII, 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 sfaII fimbrial gene cluster found in uropathogenic and newborn meningitis isolates. The sfaXII gene, located at the distal end of the sfaII 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 SfaXII 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 SfaXIIC70S mutant. The protein was produced in E. coli BL21 (DE3) cells grown in autoinduction culture media. The plasmid vector harbored DNA encoding the SfaXIIC70S 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 SfaXII can be mediated via direct binding to DNA.