Purification of the Neisseria meningitidis transferrin binding protein-2 (TBP2) to homogeneity using column chromatography.
A method for purifying TBP2 from N. meningitidis has been developed using affinity chromatography on Tf-agarose followed by ion exchange chromatography; the Tf-binding activity of fractions was evaluated by a dot-blot technique. This method allowed the purification of the TBP2 in milder conditions than those used previously and to a high degree of homogeneity as was demonstrated by Coomassie brilliant blue or Silver staining after SDS-PAGE electrophoresis. The SDS-PAGE profile of the material obtained in the Tf-agarose affinity chromatography step shows only two detectable proteins, identified as the TBP1 and the TBP2, with a small amount of contamination. Passage through a MonoQ HR anion exchange column, allowed the isolation of TBP2 in the absence of TBP1. Our results demonstrate the conservation of the antigenic reactivity of this protein, which produces monospecific serum with the antibodies elicited reacting exclusively with the TBP2 in whole outer membrane vesicles.
Ferrón
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Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Santiago de Compostela, Spain.
Ferreirós
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CM
Criado
M T
MT
Andrade
M P
MP
D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007501:Iron; D033862:Iron-Binding Proteins; D009345:Neisseria meningitidis / Q000737:chemistry*; D014168:Transferrin; D033882:Transferrin-Binding Protein B; D033863:Transferrin-Binding Proteins
D002352:Carrier Proteins; D033862:Iron-Binding Proteins; D014168:Transferrin; D033882:Transferrin-Binding Protein B; D033863:Transferrin-Binding Proteins; D007501:Iron