Improved method for preparation of lipopolysaccharide-binding protein from human serum by electrophoretic and chromatographic separation techniques.
Recent work has established the importance of serum proteins which interact with endotoxin (lipopolysaccharide, LPS) from Gram-negative bacteria. Thus human monocytes are activated after binding LPS complexed with a serum protein. LPS-binding protein (LBP) is a protein present in both normal and acute phase sera which binds LPS with high affinity. We describe the purification of LBP from human acute phase serum. The purification procedures combine preparative isoelectric focusing (IEF) and either preparative polyacrylamide gel electrophoresis (PAGE) or alternatively an anion-exchange chromatographic step using a Mono Q HR 5/5 column. This allows the isolation of biologically active LBP. LBP was characterized by N-terminal sequence analysis and by measuring the biological activity using flow cytometry (fluorescence-activated cell sorter, FACS) and a luminol enhanced chemiluminescence (LECL) assay.
Schletter
J
J
Institute of Immunology and Transfusion Medicine, Medical Faculty, Ernst-Moritz-Arndt-University, Greifswald, Germany.
Krüger
C
C
Lottspeich
F
F
Schütt
C
C
D000209:Acute-Phase Proteins; D002352:Carrier Proteins / Q000097:blood*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006801:Humans; D007525:Isoelectric Focusing; D008070:Lipopolysaccharides / Q000378:metabolism; D008163:Luminescent Measurements; D008562:Membrane Glycoproteins; D009000:Monocytes / Q000378:metabolism; D017421:Sequence Analysis; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D002352:Carrier Proteins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D000099231:Lipopolysaccharide-Binding Protein