title: High resolution clear native electrophoresis (hrCNE) allows a detailed analysis of the heterotrimeric structure of recombinant Neisseria meningitidis porins inserted into liposomes.
abstract: Three recombinant proteins of Neisseria meningitidis, rPorB, rPorA, and rRmpM, were purified and incorporated into liposomes prepared by dialysis-extrusion. The protein complexes formed using different combinations of recombinant proteins were studied by high resolution clear native electrophoresis (hrCNE) and 2-D hrCNE/SDS-PAGE, analyzing the influence of the stoichiometry of the two porins in the formation of complexes and comparing them with native porin complexes present in OMVs from five different N. meningitidis strains. Insertion of the recombinant proteins into liposomes allowed a complete refolding of porin complexes, and the electrophoretic analyses showed that, when the three recombinant proteins are present, the pattern of porin complexes obtained is similar to that observed in native OMVs. We could show homocomplexes of each individual porin and PorA/PorB, RmpM/PorB, and PorA/PorB/RmpM heterocomplexes. Our results suggest that RmpM binds only to PorB, confirm the trimeric structure of N. meningitidis pores, and demonstrate that insertion into liposomes restores the native structure of porin complexes.
authors.affiliation: Departamento de Microbiología y Parasitología, Facultad de Farmacia, Campus Sur, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
authors.forename: Paula
authors.initials: P
authors.lastname: Freixeiro
authors.forename: Ernesto
authors.initials: E
authors.lastname: Diéguez-Casal
authors.forename: Liliana
authors.initials: L
authors.lastname: Costoya
authors.forename: Juan
authors.initials: J
authors.lastname: Marzoa
authors.forename: Carlos M
authors.initials: CM
authors.lastname: Ferreirós
authors.forename: María Teresa
authors.initials: MT
authors.lastname: Criado
authors.forename: Sandra
authors.initials: S
authors.lastname: Sánchez
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008081:Liposomes / Q000737:chemistry*; D009345:Neisseria meningitidis / Q000737:chemistry*; D018272:Porins / Q000737:chemistry*; D011485:Protein Binding; D055503:Protein Multimerization; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D008081:Liposomes; D018272:Porins; D011994:Recombinant Proteins; C499670:RmpM protein, Neisseria; C057699:porin protein, Neisseria