Solubilization, isolation, and immunochemical characterization of the major outer membrane protein from Rhodopseudomonas sphaeroides.
Solubilization of the major outer membrane protein of Rhodopseudomonas sphaeroides, and subsequent isolation, has been achieved by both non-detergent- and detergent-based methods. The protein was differentially solubilized from other outer membrane proteins in 5 M guanidine thiocyanate which was exchanged by dialysis for 7 M urea. The urea-soluble protein was purified to homogeneity by a combination of DEAE-Sephadex chromatography and preparative electrophoretic techniques. Similar to the peptidoglycan-associated proteins of other Gram-negative bacteria, the protein was also purified by differential temperature extraction of the outer membrane in the presence of sodium dodecyl sulfate (SDS) followed by preparative SDS-polyacrylamide gel electrophoresis. Immunochemical analysis of the proteins isolated by the two techniques established the immunochemical identity and homogeneity of each preparation. Immunoblots of SDS-polyacrylamide gels revealed that antibody directed against the major outer membrane protein reacted with the three high molecular weight aggregates present in the outer membrane which we have previously shown to be composed of the major outer membrane protein and three nonidentical small molecular weight proteins.
D001426:Bacterial Proteins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D019791:Guanidine; D006146:Guanidines; D007158:Immunologic Techniques; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification*; D012242:Rhodobacter sphaeroides / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate; D012995:Solubility; D014508:Urea
D001426:Bacterial Proteins; D000939:Epitopes; D006146:Guanidines; D046911:Macromolecular Substances; D008565:Membrane Proteins; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D019791:Guanidine