PURIFICATION OF LIPOPOLYSACCHARIDE-BINDING PROTEIN FROM BOVINE SERUM

Citation
Pn. Bochsler et al., PURIFICATION OF LIPOPOLYSACCHARIDE-BINDING PROTEIN FROM BOVINE SERUM, Veterinary immunology and immunopathology, 51(3-4), 1996, pp. 303-314
Citations number
24
Categorie Soggetti
Immunology,"Veterinary Sciences
ISSN journal
01652427
Volume
51
Issue
3-4
Year of publication
1996
Pages
303 - 314
Database
ISI
SICI code
0165-2427(1996)51:3-4<303:POLPFB>2.0.ZU;2-T
Abstract
Lipopolysaccharide-binding protein (LBP) plays a central role in prese ntation of bacterial-derived lipopolysaccharide (LPS; endotoxin) to le ukocytes such as macrophages and neutrophils, Interaction of LBP with LPS is significant because LBP-LPS complexes promote activation of leu kocytes and the immune system, which results in enhanced secretion of a spectrum of proinflammatory cytokines. An improved, simplified metho d was used to purify bovine LBP from serum, Methodology consisted of i on-exchange chromatography using Bio-Rex 70 resin, followed by gel-fil tration chromatography (Sephacryl S-200 resin) of a selected ion-excha nge 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 64 kD protein (bovine LBP) in the cleanest fractions. The isoelectric point of bovine LBP was determine d to be 6.8. Identity of the protein was substantiated by western-blot analysis, and by N-terminus amino acid sequence analysis with favorab le comparison to published sequence data from rabbit,human, and murine LBP Identity was corroborated by use of purified bovine LBP in bioass ays which demonstrated enhanced tissue factor expression of LPS (1 ng ml(-1))-stimulated bovine alveolar macrophages, Tissue factor expressi on was inhibitable in these assays using anti-CD14 monoclonal antibodi es, which is also consistent with LBP-mediated activation of cells. Wh en bovine LBP was heated at 56 degrees C for 30 min, the biological ac tivity was reduced by 50% in the macrophage-based bioassays. Biologica l activity of bovine LBP was completely destroyed by heating at 62 deg rees C for 30 min, which compared favorably with data resulting from u se of fetal bovine serum.