Purification and characterization of human soluble CD14 expressed in Pichia pastoris.
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.
Nomura
Shin-ichi
S
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
Inamori
Kei-ichiro
K
Muta
Tatsushi
T
Yamazaki
Soh
S
Sunakawa
Yuko
Y
Iwanaga
Sadaaki
S
Takeshige
Koichiro
K
D015153:Blotting, Western; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D011994:Recombinant Proteins