Expression and refolding of functional fragments of the human lipopolysaccharide receptor CD14 in Escherichia coli and Pichia pastoris.
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.
Majerle
A
A
Laboratory for Molecular Modeling and NMR Spectroscopy, National Institute of Chemistry, Hajdrihova 19, Ljubljana, 1000, Slovenia.
Kidric
J
J
Jerala
R
R
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D018950:Lipopolysaccharide Receptors / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D018950:Lipopolysaccharide Receptors; D010446:Peptide Fragments; D011994:Recombinant Proteins