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CD14, a 55kDa lipopolysaccharide binding glycoprotein, is a key element in both LPS-mediated activation of cells and endotoxin detoxification. A gene fragment containing residues 1-348 of the human LPS receptor CD14, representing the extracellular form of the molecule, was fused to the CH(2)-CH(3) portion of the human IgG heavy chain or to a 6x His tag and transfected into CHO cells. Stable cell lines of each were grown to produce recombinant protein in unsupplemented serum free media and CD14His was purified by ion-exchange chromatography. After passive immobilization onto a carbon surface both forms of the CD14 fusion proteins bound LPS-biotin in a dose-dependent manner in an electrochemiluminescent assay. Binding was inhibited by the anti-CD14 antibody S39 as well as by unlabeled LPS. This report describes an efficient method of purifying CD14 and a novel assay to detect bioactive lipopolysaccharide.

CD14 is a protein that mediates lipopolysaccharide (LPS)-induced biological responses such as activation of a transcriptional factor, nuclear factor (NF)-kappaB. It exists as a soluble form (sCD14) in serum and mediates LPS responses of epithelial and endothelial cells as well as a membrane-bound form (mCD14) on monocytes and macrophages. To obtain sCD14 in large quantity for its structural and functional characterization, we expressed the full-length form of human recombinant sCD14 (rsCD14) in a methylotrophic yeast, Pichia pastoris. The recombinant protein was expressed as a major protein in the culture supernatant and purified by ammonium sulfate precipitation, followed by three steps of ion exchange chromatographies. Finally, 1.6 mg of the protein was obtained in high purity from 2L of the supernatant and its identity to sCD14 was confirmed by NH(2)-terminal amino acid sequence analysis. The purified protein was found to have N-linked sugars by an analysis of enzymatic deglycosylation. A native PAGE analysis revealed that the protein was able to form complexes with LPS. In addition, the rsCD14 protein could mediate the LPS-mediated activation of NF-kappaB in human embryonic kidney 293 cells transfected with Toll-like receptor 4 and MD-2, indicating that the purified protein is biologically active. Thus, the rsCD14 protein expressed in P. pastoris and highly purified in a large amount is useful for its structural and functional studies.

CD14 is a high-affinity cellular receptor specific for bacterial lipopolysaccharides (LPS), present in the bacterial cell wall. Binding of LPS to CD14 initiates the innate component of immune response and triggers a response that can lead to septic shock. In order to provide recombinant protein for the study of LPS-CD14 molecular interactions we have expressed human CD14 in Escherichia coli and Pichia pastoris. In bacteria, the protein was produced in high yield in the form of inclusion bodies. We have optimized the procedure for its refolding and generated correctly folded protein. A procedure to monitor the refolding efficiency by using conformation-specific anti-human CD14 monoclonal antibody has been established. A fragment of 152 amino acids of CD14 which retains the ability to bind LPS has been produced in a methylotrophic yeast, P. pastoris, expression system. The recombinant protein from yeast is glycosylated and secreted into the medium. The CD14 fragment was purified to homogeneity by immunoaffinity chromatography. Recombinant CD14 from both bacteria and yeast bind to LPS., (Copyright 1999 Academic Press.)