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Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction
Published on: May 28, 2012
Purification of lipopolysaccharide-binding protein from bovine serum
P N Bochsler1, Z Yang, C L Murphy
1Department of Pathology, College of Veterinary Medicine, University of Tennessee, Knoxville 37901, USA.
Insights
Researchers developed a simplified method to purify bovine lipopolysaccharide-binding protein (LBP), crucial for immune responses to bacterial endotoxins. The purified LBP enhanced immune cell activity, confirming its role in innate immunity.
Area of Science:
- Immunology
- Biochemistry
Background:
- Lipopolysaccharide-binding protein (LBP) facilitates the immune response to bacterial lipopolysaccharide (LPS) by presenting LPS to leukocytes.
- LBP-LPS complexes activate immune cells, leading to the release of pro-inflammatory cytokines.
Purpose of the Study:
- To develop an improved and simplified method for purifying bovine LBP from serum.
- To characterize the purified bovine LBP and confirm its biological activity.
Main Methods:
- Ion-exchange chromatography (Bio-Rex 70) followed by gel-filtration chromatography (Sephacryl S-200).
- Purity assessed by SDS-PAGE and densitometry.
- Identity confirmed by Western blot and N-terminal amino acid sequencing.
- Biological activity evaluated through macrophage-based bioassays measuring tissue factor expression.
Main Results:
- Achieved up to 88.7% purity of a 64 kDa bovine LBP.
- Determined the isoelectric point of bovine LBP to be 6.8.
- Confirmed biological activity through enhanced LPS-stimulated tissue factor expression in macrophages, inhibitable by anti-CD14 antibodies.
- Demonstrated that heating bovine LBP at 56°C for 30 min reduced activity by 50%, and at 62°C for 30 min completely abolished it.
Conclusions:
- A simplified purification method for bovine LBP was successfully established.
- The purified bovine LBP is biologically active and plays a role in LPS-mediated immune cell activation.
- The thermal lability of bovine LBP's biological activity was characterized.
Abstract:
Lipopolysaccharide-binding protein (LBP) plays a central role in presentation of bacterial-derived lipopolysaccharide (LPS; endotoxin) to leukocytes such as macrophages and neutrophils. Interaction of LBP with LPS is significant because LBP-LPS complexes promote activation of leukocytes and the immune system, which results in enhanced secretion of a spectrum of proinflammatory cytokines. An improved, simplified method was used to purify bovine LBP from serum. Methodology consisted of ion-exchange chromatography using Bio-Rex 70 resin, followed by gel-filtration chromatography (Sephacryl S-200 resin) of a selected ion-exchange fraction (0.22-0.50 M NaCl). Densitometric scans on silver-stained polyacrylamide gels of chromatographically-derived proteins indicated up to 88.7% purity of the resultant 64kD protein (bovine LBP) in the cleanest fractions. The isoelectric point of bovine LBP was determined to be 6.8. Identity of the protein was substantiated by western-blot analysis, and by N-terminus amino acid sequence analysis with favorable comparison to published sequence data from rabbit, human, and murine LBP Identity was corroborated by use of purified bovine LBP in bioassays which demonstrated enhanced tissue factor expression of LPS (1 ng ml(-1)-stimulated bovine alveolar macrophages. Tissue factor expression was inhibitable in these assays using anti-CD14 monoclonal antibodies, which is also consistent with LBP-mediated activation of cells. When bovine LBP was heated at 56 degrees C for 30 min, the biological activity was reduced by 50% in the macrophage-based bioassays. Biological activity of bovine LBP was completely destroyed by heating at 62 degrees C for 30 min, which compared favorably with data resulting from use of fetal bovine serum.
