Purification and characterization of human soluble CD14 expressed in Pichia pastoris

Shin Ichi Nomura, Kei Ichiro Inamori, Tatsushi Muta, Soh Yamazaki, Yuko Sunakawa, Sadaaki Iwanaga, Koichiro Takeshige

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

CD14 is a protein that mediates lipopolysaccharide (LPS)-induced biological responses such as activation of a transcriptional factor, nuclear factor (NF)-κB. 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 2 L of the supernatant and its identity to sCD14 was confirmed by NH2-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-κB 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.

Original languageEnglish
Pages (from-to)310-320
Number of pages11
JournalProtein Expression and Purification
Volume28
Issue number2
DOIs
Publication statusPublished - 2003 Apr 1

ASJC Scopus subject areas

  • Biotechnology

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