title: High-level expression of soluble form of mouse natural killer cell receptor NKR-P1C(B6) in Escherichia coli.
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.
authors.affiliation: Department of Biochemistry, Charles University in Prague, CZ-12840 Prague 2, Czech Republic.
authors.forename: Daniel
authors.initials: D
authors.lastname: Rozbeský
authors.forename: Daniel
authors.initials: D
authors.lastname: Kavan
authors.forename: Josef
authors.initials: J
authors.lastname: Chmelík
authors.forename: Petr
authors.initials: P
authors.lastname: Novák
authors.forename: Ondřej
authors.initials: O
authors.lastname: Vaněk
authors.forename: Karel
authors.initials: K
authors.lastname: Bezouška
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism*; D007694:Killer Cells, Natural / Q000276:immunology / Q000378:metabolism; D008024:Ligands; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D055651:NK Cell Lectin-Like Receptor Subfamily B / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
chemical_list: D000954:Antigens, Surface; D008024:Ligands; D055651:NK Cell Lectin-Like Receptor Subfamily B; D011994:Recombinant Proteins