Solubilization, isolation, and immunochemical characterization of the major outer membrane protein from Rhodopseudomonas sphaeroides.

C D Deal,S Kaplan

doi:10.1016/s0021-9258(18)32443-8

Abstract

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.

Highlights

sodium dodecyl sulfate (SDS)-polyacrylamidgeellectrophoresiIsm. munoof the aggregates together with its abundance would suggest chemical analysis of the proteins isolated by the two an important role for it in the outer membrane
Rected against the major outer membrane protein re- Studies involving the intracytoplasmic membrane proteins acted with the three high molecular weiagghgtregates of R. sphaeroides by Cohen and Kaplan [21] have demonpresent in the outer membrane which we have previ- strated theapplicability of disrupting the membrane with the ously shown to be composed of the major outer mem- chaotropic salt guanidine thiocyanate as initially described by brane protein and three nonidentical small molecular Moldow et al
Againstconcentratedurea solutions, to obtainproteinsin solubilized aqueous form, which are thenamenable tofurther purification procedures. This technique was suggested to be Studies in many of the Gram-negative bacteria have re- of general use in the isolation of membrane proteins. In this vealed the outermembrane tobe a complexstructure involved paper, we report the application of this method to theisolation in maintainingthe structuralintegrity of the cell (l), of the major outer membrane protein of R. sphaeroides

Summary

AND RESULTS”’

Lipoproteins [17,18,19]. Previous studies inour laboratory [20] have reported on the initial characterization of the isolated outer membrane of Rhodopseudomonassphaeroides from chemoheterotrophi-.

DISCUSSION

MATERIALS AND METHODS

II GuSCN extracted-urea SOlUhle protein