Identification of Neisseria meningitidis outer membrane vesicle complexes using 2-D high resolution clear native/SDS-PAGE.
The identification and characterization of meningococcal outer membrane vesicle complexes can be important for gaining an in-depth understaining of their structure and functionality. Analysis of the vesicle complexome by 'traditional' 2-D analysis, in which isoelectrofocusing is used for separation in the first dimension, is hampered by the high hydrophobicity and extreme isoelectric points of many relevant proteins. Analysis of the meningococcal outer membrane vesicle complexome using Blue Native (nondenaturing) electrophoresis instead of isoelectrofocusing in the first dimension showed several porin complexes, but their composition could not be clearly resolved after separation by SDS-PAGE in the second dimension. In this work, using a recently described native separation technique -high resolution Clear Native Electrophoresis-and different bidimensional approaches, we were able to demonstrate the presence of relevant outer membrane complexes which could be resolved with a higher resolution than in previous analysis. The most relevant were nine porin complexes formed by different combinations of the meningococcal PorA, PorB and RmpM proteins, and comparison with the complexes formed in specific knockout mutants allowed us to infer the relevance of each porin in the formation of each complex.
Marzoa
Juan
J
Departamento de Microbiología y Parasitología, Facultad de Farmacia, Campus Sur, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
Sánchez
Sandra
S
Ferreirós
Carlos M
CM
Criado
María Teresa
MT
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D009345:Neisseria meningitidis / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001425:Bacterial Outer Membrane Proteins