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High-level Expression of Soluble Form of Mouse Natural Killer Cell Receptor NKR-P1C(B6) in Escherichia Coli

High-level Expression of Soluble Form of Mouse Natural Killer Cell Receptor NKR-P1C(B6) in Escherichia Coli

Daniel Rozbeský, Daniel Kavan, Josef Chmelík, Petr Novák, Ondřej Vaněk, Karel Bezouška

Protein Expr Purif. 2011 Jun;77(2):178-84.

PMID: 21284957

Abstract:

Mouse NKR-P1C(B6) receptor corresponding to NK1.1 alloantigen is one of the most widespread surface markers of mouse NK and NKT cells in C57BL/6 mice detected by monoclonal antibody PK136. Although functional studies revealed the ability of this receptor to activate both natural killing and production of cytokines upon antibody crosslinking, the ligand for NKR-P1C(B6) remains unknown. In order to initiate ligand identification, structural studies, and epitope mapping experiments, we developed a simple and efficient expression and purification protocol allowing to produce large amounts of pure soluble monomeric mouse NKR-P1C(B6). Our protein encompassed approximately half of the stalk region and the entire C-terminal globular ligand binding domain. The identity of protein that was devoid of N-terminal initiation methionine and had all three expected disulfides closed was confirmed using high resolution ion cyclotron resonance mass spectrometry. Protein produced into inclusion bodies in Escherichia coli was efficiently refolded into a unique three dimensional structure as confirmed by NMR using (1)H-(15)N-HSQC spectra of uniformly labeled protein. The exceptional purity of the protein should allow its crystallization and detailed structural investigations, and is a prerequisite for its use as a probe in ligand identification and antibody epitope mapping experiments.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR4246983	Monoclonal Anti-S1P4, C-Terminal antibody produced in mouse			Price

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