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 outermembrane 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.

A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles

“…Especially P1, P2, and P5 seem to be highly enriched in sOM compared to dgOM preparations. Although OM purification using sarkosyl treatment is usually the first choice method in terms of purity, it has also been published that the number of proteins in those preparations is significantly reduced possibly due to excessive washing steps required for this method (Hobb et al, 2009). We believe that this may be also true in our case, where some OMPs might get washed out, whereas especially the ˇ-barrel proteins like P1, P2, and P5 seem to remain in the OM sample leading to an artificial enrichment of these kinds of proteins.…”

Cellular solid-state nuclear magnetic resonance spectroscopy

“…The CE fraction was isolated by ultracentrifugation of the cell lysate for 8 min at 150;000 × g and at 4°C. The correct localization and membrane insertion of PagL was confirmed on Coomassie-stained SDS/ PAGE gels after extracting the CE with 1% N-lauroylsarcosine or 6 M urea as described previously (29,30). To obtain the reference PL ssNMR spectra, PagL was expressed as intracellular inclusion bodies in E. coli BL21Star(DE3) harboring the plasmid pPagL ðPaÞ (-), then purified, and reconstituted in dimyristoylphosphatidylcholine vesicles at a final protein-to-lipid molar ratio of 1∶50 as described in SI Materials and Methods.…”

Proteomics for development of vaccine

“…Such observations indicate that for each pathogenic species several methods for protein isolation and enrichment of particular protein categories have to be experimentally checked to find the one that is most efficient and compatible with further analysis. Such a comprehensive study of C. jejuni OMPs has recently been published [66].…”