title: Pseudomonas aeruginosa outer membrane protein OprF as an expression vector for foreign epitopes: the effects of positioning and length on the antigenicity of the epitope.
abstract: OprF, the major outer membrane (OM) protein of Pseudomonas aeruginosa, has been proposed to be comprised of a series of beta-strands separated by periplasmic or surface-exposed loop regions. In this study, a simple malarial epitope was used to demonstrate that OprF can be used as an expression vector to present foreign peptide sequences, namely, the 4-amino-acid (aa) repeating epitope (Asn-Ala-Asn-Pro = NANP) of the circumsporozoite protein of the human malarial parasite Plasmodium falciparum. Eight permissive sites, that allowed the expression and surface exposure of the malarial epitope, were identified throughout OprF. Using a monoclonal antibody (mAb) specific for the malarial epitope, we investigated the effects of positioning and length of the epitope on its antigenicity in the OprF expression vector system. It was demonstrated that the malarial epitope inserted at aa26 was significantly more reactive with the epitope-specific mAb (i.e., more antigenic) when assayed in the context of whole cells whereas those at aa213 and aa290 were more antigenic when assayed in the OM. The malarial epitope inserted at aa188 and aa196 was moderately antigenic, while this epitope inserted at aa215 and aa310 showed low antigenicity with the same mAb in both whole cell and OM assays. For two insertion sites, aa26 and aa213, we demonstrated that the insertion of multiple copies of the epitope enhanced reactivity with the malarial epitope-specific mAb. These data are discussed with respect to the local OprF sequences into which the epitope was inserted.
authors.affiliation: Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
authors.forename: R S
authors.initials: RS
authors.lastname: Wong
authors.forename: R A
authors.initials: RA
authors.lastname: Wirtz
authors.forename: R E
authors.initials: RE
authors.lastname: Hancock
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D000939:Epitopes / Q000235:genetics* / Q000276:immunology; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010963:Plasmodium falciparum / Q000235:genetics / Q000276:immunology; D018272:Porins / Q000235:genetics* / Q000276:immunology; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000939:Epitopes; D009838:Oligodeoxyribonucleotides; D018272:Porins; D011993:Recombinant Fusion Proteins