title: Evaluation of procedures for outer membrane isolation from Campylobacter jejuni.
abstract: Although infection with Campylobacter jejuni is one of the leading causes of gastroenteritis worldwide, relatively little is known about the factors that are required to elicit a protective immune response. The need for a vaccine against this pathogen is well recognized and a number of vaccine candidates have been tested with varying degrees of success; however, there is still a lack of a suitable vaccine. To gain a better understanding of the outer-membrane protein components of this organism, a 'gold standard' method to purify the outer membrane is needed. Therefore, we attempted to develop a robust and reliable method which resulted in a pure outer-membrane fraction. A total of nine methodologies were examined and analysed by SDS-PAGE and immunoblotting using subcellular markers for the cytoplasm, cytoplasmic membrane and outer membrane. We found that glycine extraction, differential detergent extraction using Triton X-100, serial extraction using 1 M Tris pH 7, spheroplasting by lysozyme and sonication, and carbonate extraction did not produce pure outer-membrane preparations. However, we identified three methods that provided outer-membrane fractions free from subcellular contamination. Isopycnic centrifugation using a 30-60 % sucrose gradient produced seven fractions free from cytoplasmic or cytoplasmic membrane contamination; however, these fractions did not correspond as well as expected with the typical outer-membrane-associated peak (e.g. Escherichia coli or Salmonella). The spheroplast method using lysozyme alone also resulted in pure outer-membrane fraction, as did carbonate washing of this sample. The extraction of outer membranes using N-lauroylsarcosine (Sarkosyl) produced the purest and most reproducible sample. These outer-membrane preparations will be useful for future studies aimed at identifying C. jejuni surface proteins as vaccine components.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA.
authors.forename: Rhonda I
authors.initials: RI
authors.lastname: Hobb
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Fields
authors.affiliation: College of Biomedical Science, Florida Atlantic University, Boca Raton, FL 33431, USA.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Burns
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA.
authors.forename: Stuart A
authors.initials: SA
authors.lastname: Thompson
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D016123:Campylobacter jejuni / Q000737:chemistry*; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D017830:Octoxynol / Q000737:chemistry; D015203:Reproducibility of Results; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000737:chemistry; D012680:Sensitivity and Specificity
chemical_list: D001425:Bacterial Outer Membrane Proteins; C025231:sarkosyl; D017830:Octoxynol; D012521:Sarcosine