Lipopolysaccharide binding protein from normal human plasma purified with high efficiency.
Lipopolysaccharide (LPS)-binding protein (LBP) is an acute-phase plasma glycoprotein of 60 kDa which functions as an opsonin on activation of macrophages by bacterial LPS. The importance of LBP on the host defense against infection has been demonstrated. Human LBP has been purified from ascitic fluid or acute-phase serum. However, clinical samples often are not available, so we developed a method to purify LBP from normal plasma. The purification was accomplished by barium citrate precipitation, followed by three-step ion-exchange chromatography. The final step was Mono S cation-exchange chromatography, following Bio-Rex 70 and Mono Q chromatography, and it enabled the specific activity to increase overall 48,000-fold. This seems to be the first report of the use of Mono S column to purify LBP. The LBP obtained using these steps proved to be homogeneous by SDS-PAGE, Western blotting, and N-terminal sequence and amino acid composition analyses. The purification method established here will compensate when normal plasma with a low LBP content is available as the starting source.
Nanbo
A
A
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Nishimura
H
H
Nagasawa
S
S
D000209:Acute-Phase Proteins / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D000837:Anion Exchange Resins; D001466:Barium Sulfate; D016466:CHO Cells; D002352:Carrier Proteins / Q000097:blood*; D002411:Cation Exchange Resins; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007475:Ion Exchange Resins; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012117:Resins, Synthetic; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D000837:Anion Exchange Resins; D001466:Barium Sulfate; D002352:Carrier Proteins; D002411:Cation Exchange Resins; D007475:Ion Exchange Resins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D012117:Resins, Synthetic; D000099231:Lipopolysaccharide-Binding Protein; C106553:Mono-S; C042855:Mono Q; C026257:Bio-Rex 70