Linked Life Data

12699696

Source:http://linkedlifedata.com/resource/pubmed/id/12699696

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2003-4-17
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
1046-5928
pubmed:author
pubmed:issnType
Print
pubmed:volume
28
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
310-20
pubmed:dateRevised
2006-4-21
pubmed:meshHeading
pubmed:year
2003
pubmed:articleTitle
Purification and characterization of human soluble CD14 expressed in Pichia pastoris.
pubmed:affiliation
Department of Molecular and Cellular Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. nomura-s@biochem2.med.kyushu-u.ac.jp
pubmed:publicationType
Journal Article