Expression and purification of the recombinant enteropathogenic Escherichia coli vaccine candidates BfpA and EspB.
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 NH(2)-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.
Quintana Flores
Victor Martin
VM
Universidade Estadual do Norte Fluminense, Rio de Janiero, Brazil.
Campos de Souza Fernandes
Regina Célia
RC
Sousa de Macedo
Zila
Z
Medina-Acosta
Enrique
E
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001428:Bacterial Vaccines / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism* / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000235:genetics*; D036781:Fimbriae Proteins / Q000235:genetics*; D006801:Humans; D007070:Immunoglobulin A / Q000378:metabolism; D008756:Methylgalactosides / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D012475:Salmonella / Q000378:metabolism; D013862:Thiogalactosides / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; C104901:BfpA protein, E coli; C087863:EaeB protein, E coli; D029968:Escherichia coli Proteins; D007070:Immunoglobulin A; D008756:Methylgalactosides; D011994:Recombinant Proteins; D013862:Thiogalactosides; D036781:Fimbriae Proteins; C009468:thiomethylgalactoside