Biophysical and antigenic characterization of gonococcal protein I incorporated into liposomes.
The major gonococcal outer membrane protein, protein I (Por), was reconstituted into liposomes composed of either 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC) or POPC:1-palmitoyl, 2-oleoyl phosphatidylethanolamine (POPE) (1:1 weight ratio) and the resulting proteoliposomes characterized with respect to their biophysical and antigenic properties. Isopycnic density gradient centrifugation studies established that essentially all of the protein was reconstituted into the lipid bilayer with no significant differences in incorporation seen as a function of lipid composition. Examination of Por orientation in these proteoliposomes revealed that over 80% of the protein was oriented facing outwards in the same 'hairpin loop' fashion found in the native bacterial membrane. Reconstituted Por proteoliposomes exhibited a mean vesicle diameter of > 0.5 micron but could be reduced by extrusion without significant loss of protein or lipid. These extruded systems were suitable for sterilization by terminal filtration. The antibody binding activities of various Por liposome formulations were determined using both anti-Por monoclonal antibodies and an immunized rabbit sera. No significant differences in antibody binding were observed as a function of proteoliposome lipid composition. However, consistently higher levels of antibody binding were obtained for Por liposomes prepared in this way compared with reconstituted systems prepared as described in earlier publications.
Parmar
M M
MM
Department of Pharmacology and Therapeutics, University of British Columbia, Vancouver, Canada.
Blake
M S
MS
Madden
T D
TD
D000818:Animals; D000907:Antibodies, Bacterial / Q000378:metabolism; D001428:Bacterial Vaccines / Q000008:administration & dosage*; D002499:Centrifugation, Density Gradient; D004797:Enzyme-Linked Immunosorbent Assay; D008081:Liposomes; D051379:Mice; D009344:Neisseria gonorrhoeae / Q000276:immunology*; D018272:Porins / Q000008:administration & dosage* / Q000276:immunology; D011817:Rabbits
D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D008081:Liposomes; D018272:Porins; C057699:porin protein, Neisseria