Pn. Bochsler et al., PURIFICATION OF LIPOPOLYSACCHARIDE-BINDING PROTEIN FROM BOVINE SERUM, Veterinary immunology and immunopathology, 51(3-4), 1996, pp. 303-314
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.