BfpA, the structural repeating protein subunit A of the bundle-forming pilus and EspB, a type-III-secreted pore-forming protein of enteropathogenic Escherichia coli (EPEC), both virulence factors central for EPEC pathogenesis, were overexpressed in E. coli DH5alpha and M15 laboratory strains, respectively, using the pQE-30 cloning expression system, as chimeric fusions to a NH2-terminal histidine hexapeptide (His(6)-tag) sequence. After isopropyl,beta-D-thiogalactoside induction, the expression levels achieved were 11 and 40% of total soluble protein for BfpA and EspB, respectively. The His(6)-tagged recombinant proteins were purified (up to 98% homogeneity) by Ni-agarose affinity chromatography and produced yields varying from 0.65 to 3.1 mg of recombinant protein per gram of wet weight cells. The immunogenicity and antigenicity of the final products were tested in rabbits and using fecal specimens obtained from children suffering from acute watery diarrhea, respectively. The recombinant products correspond to antigenically authentic protein standards, useful in future epidemiological and neonatal vaccinology studies. (C) 2002 Elsevier Science (USA).