High resolution clear native electrophoresis is a good alternative to blue native electrophoresis for the characterization of the Escherichia coli membrane complexes.

Blue native electrophoresis (BNE) has become the most popular method for the global analysis of membrane protein complexes. Although it has been shown to be very useful for that purpose, it can produce the dissociation of complexes with weak interactions and, due to the use of Coomassie Brilliant Blue, does not allow the subsequent application of fluorimetric and/or enzymatic techniques. Recently, we have successfully used the high resolution clear native electrophoresis (hrCNE) for the analysis of Neisseria meningitidis outer membrane porin complexes. The aim of this study was to determine the composition of the complexome of the Escherichia coli envelope by using hrCNE and to compare our results with those previously obtained using BNE. The bidimensional electrophoresis approaches used, hrCN/hrCNE and hrCN/SDS-PAGE, coupled to mass spectrometry allowed a detailed analysis of the complexome of E. coli membranes. For the first time, the three subunits of the formate dehydrogenase FDH-O were identified forming a single complex and hrCNE also allowed the identification of both the HflK and HflC proteins as components of the HflA complex. This technique also allowed us to suggest a relationship between OmpF and DLDH and, although OmpA is considered to be monomeric in vivo, we found this protein structured as homodimers. Thus hrCNE provides a good tool for future analyses of bacterial membrane proteins and complexes and is an important alternative to the commonly used BNE.

Study of the stability of proteoliposomes as vehicles for vaccines against Neisseria meningitidis based on recombinant porin complexes.

Although effective against epidemic serogroup B Neisseria meningitidis strains, vaccines based on outer membrane vesicles continue to present important limitations, and great efforts are currently being focused in the development of a variety of new vaccine candidates and in the reformulation of currently existing ones. In this work, three N. meningitidis proteins, the PorA and PorB porins and the RmpM protein, were cloned, purified and incorporated into liposomes to build defined systems. The ability of proteoliposomes to allow the refolding porin complexes, and their stability during storage at 4°C and after lyophilization in presence of two cryoprotection agents, glucose and trehalose, were evaluated. This approach allowed to mimic the porin complexes present in natural OMVs, reducing the content of hypervariable protein PorA. During storage at 4°C, our systems showed some changes in the morphology and aggregation after three months, while after lyophilization the systems maintained their properties during the whole nine months of storage checked, with glucose allowing the best preservation of the antigenic properties of the proteins in the proteoliposomes.

High resolution clear native electrophoresis (hrCNE) allows a detailed analysis of the heterotrimeric structure of recombinant Neisseria meningitidis porins inserted into liposomes.

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.