Work

Evaluating size exclusion chromatography for nucleic acid removal in Klebsiella pneumoniae cell surface polysaccharide purification

Year: 2025

Type: article

Abstract: Cell surface-associated polysaccharides in Klebsiella are major virulence determinants and crucial targets for developing vaccines. Traditionally, the purification of these cell surface polysaccharides from Klebsiella pneumoniae involves a multi-step process comprising phenol extraction , nuclease digestion, ultracentrifugation , and repeated ethanol extractions. In this study, we evaluated size exclusion chromatography for effectively eliminating nucleic acid contamination while purifying high molecular weight cell surface-associated polysaccharides . Post-initial extraction, the nucleic acid content remains significantly elevated, and kinetic analysis reveals that DNase I and RNase A digestion is neither economically viable nor effective for removing these contaminants. Employing an appropriate size exclusion resin removes over 99 % of nucleic acid contamination, as confirmed by nucleic acid content analysis and agarose gel electrophoresis . Purity and structural analysis using 1 H 1D-NMR and 2D-NMR demonstrate that the cell surface-associated polysaccharide purified with this study is highly homogeneous and identified as antigenic O-polysaccharide. This approach streamlines the purification process by eliminating the need for nuclease digestion and additional ethanol precipitation steps.

Source: Carbohydrate Polymers

DOI: 10.1016/j.carbpol.2025.123531