Preparation and characterization of truncated human lipopolysaccharide-binding protein in Escherichia coli

• Published in: Protein expression and purification 2006-10, Vol.49 (2), p.276-283
• Main Authors: Kohara, Jun, Tsuneyoshi, Naoko, Gauchat, Jean-François, Kimoto, Masao, Fukudome, Kenji
• Format: Article
• Language: English
• Subjects: Acute-Phase Proteins - chemistry; Acute-Phase Proteins - genetics; Acute-Phase Proteins - isolation & purification; Acute-Phase Proteins - metabolism; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; Escherichia coli; Escherichia coli - genetics; Humans; LBP; Lipid A - chemistry; Lipid A - metabolism; Lipopolysaccharide; Lipopolysaccharide Receptors - metabolism; Lymphocyte Antigen 96 - metabolism; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - genetics; Membrane Glycoproteins - isolation & purification; Membrane Glycoproteins - metabolism; O Antigens - chemistry; O Antigens - metabolism; Protein Binding; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Thioredoxin; Toll-Like Receptor 4 - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2006.05.015

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria, and is the causative agent of endotoxin shock. LPS induces signal transduction in immune cells when it is recognized by the cell surface complex of toll-like receptor 4 (TLR4) and MD-2. The complex recognizes the lipid A structure in LPS, which is buried in the membrane of the outer envelope. To present the Lipid A structure to the TLR4/MD-2, processing of LPS by LPS-binding protein (LBP) and CD14 is required. In previous studies, we expressed recombinant proteins of human MD-2 and CD14 as fusion proteins with thioredoxin in Escherichia coli, and demonstrated their specific binding abilities to LPS. In this study, we prepared a recombinant fusion protein containing 212 amino terminal residues of human LBP (HLB212) by using the same expression system. The recombinant protein expressed in E. coli was purified as a complex form with host LPS. The binding was not affected by high concentrations of salt, but was prevented by low concentrations of various detergents. Both rough-type LPS lacking the O antigen and smooth-type LPS with the antigen bound to HLBP212. Therefore, oligosaccharide repeats appeared to be unnecessary for the binding. A nonpathogenic penta-acylated LPS also bound to HLBP212, but the binding was weaker than that of the wild type. The hydrophobic interaction between the LBP and acyl chains of lipid A appears to be important for the binding. The recombinant proteins of LPS-binding molecules would be useful for analyzing the defense mechanism against infections.