Isolation of smooth-type lipopolysaccharides to electrophoretic homogeneity

Abstract:

The high structural heterogeneity of smooth-type lipopolysaccharides (LPS) enormously complicates the isolation of their constituent molecular species. Proof of concept is given here on the feasibility of using preparative slab-PAGE to isolate highly homogeneous smooth-type LPS glycoforms. LPS species (from 3.6 to 14.2 kDa) from Escherichia coli K-235 were separated by preparative slab-PAGE and recovered by utilizing the combined on-gel LPS reverse staining, extrusion, and passive elution techniques. As a result, 15 electro-phoretically pure LPS ftactions were obtained. The LPS content in the recovered fractions ranged from 280 ng (intermediate mobility glycoforms) to 411 μg (highest mobility glyco-forms). The quantities of LPS fractions were sufficient to allow quantitation of the Limulus amebocyte lysate (LAL) activities of these distinct-molecular-mass LPS species, in the range from (1.1 ± 0.1) × 103 to (8.7 ± 0.3) × 105 endotoxin units (EU)/mL, by standard LAL assay. We have thus definitively demonstrated that slab-PAGE may be a suitable platform to more selectively purify individual glycoform fractions from smooth-type LPS. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Año de publicación:

2007

Keywords:

• biological activity
• LIPOPOLYSACCHARIDE
• Limulus amebocyte lysate

Fuente:

scopus