ABSTRACT
Human natural killer receptor protein 1 (NKR-P1, CD161, gene klrb1) is a C-type lectin-like receptor of natural killer (NK) cells responsible for recognition of its cognate protein ligand lectin-like transcript 1 (LLT1). NKR-P1 is the single human orthologue of the prototypical rodent NKR-P1 receptors. Naturally, human NKR-P1 is expressed on the surface of NK cells, where it serves as inhibitory receptor; and on T and NKT cells functioning as co-stimulatory receptor promoting secretion of IFNγ. Most notably, it is expressed on Th17 and Tc17 lymphocytes where presumably promotes targeting into LLT1 expressing immunologically privileged niches. We tested effect of different protein tags (SUMO, TRX, GST, MsyB) on expression of soluble NKR-P1 in E. coli. Then we optimized the expression construct of soluble NKR-P1 by preparing a library of expression constructs in pOPING vector containing the extracellular lectin-like domain with different length of the putative N-terminal stalk region and tested its expression in Sf9 and HEK293 cells. Finally, a high-level expression of soluble NKR-P1 was achieved by stable expression in suspension-adapted HEK293S GnTI- cells utilizing pOPINGTTneo expression vector. Purified soluble NKR-P1 is homogeneous, deglycosylatable, crystallizable and monomeric in solution, as shown by size-exclusion chromatography, multi-angle light scattering and analytical ultracentrifugation.
Subject(s)
Killer Cells, Natural/metabolism , NK Cell Lectin-Like Receptor Subfamily B/biosynthesis , NK Cell Lectin-Like Receptor Subfamily B/isolation & purification , Bioreactors , Escherichia coli/genetics , HEK293 Cells , Humans , Lectins, C-Type/metabolism , Ligands , NK Cell Lectin-Like Receptor Subfamily B/genetics , Receptors, Cell Surface/metabolism , Th17 Cells/metabolismABSTRACT
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.
Subject(s)
Antigens, Surface/metabolism , Inclusion Bodies/metabolism , NK Cell Lectin-Like Receptor Subfamily B/metabolism , Recombinant Proteins/metabolism , Amino Acid Sequence , Animals , Antigens, Surface/genetics , Antigens, Surface/immunology , Antigens, Surface/isolation & purification , Binding Sites , Cloning, Molecular , Escherichia coli , Gene Expression , Inclusion Bodies/genetics , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Ligands , Mass Spectrometry , Mice , Mice, Inbred C57BL , Molecular Sequence Data , NK Cell Lectin-Like Receptor Subfamily B/genetics , NK Cell Lectin-Like Receptor Subfamily B/immunology , NK Cell Lectin-Like Receptor Subfamily B/isolation & purification , Protein Binding , Protein Refolding , Protein Structure, Secondary , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , SolubilityABSTRACT
The proteins on lymphocyte surface play important roles in a wide range of immunological processes, but the profile and characterization of surface proteins remain to be further investigated, among which the method for fast screening of surface proteins needs to be established. In this study, a conventional cDNA clone library of hepatic lymphocytes from C57BL/6 mouse was constructed, and then the cDNA was inserted into a recombinant expression vector pSecTag-attR with a signal peptide and tag protein for fluorescence screening. The recombinant cDNA expression library was transfected into COS-1 cells, and the transfected cells with the expressed membrane proteins were labeled by fluorescence antibodies and isolated by fluorescence activated cell sorting. After two cycles of sorting, the purity of fluorescence positive cells with membrane proteins was up to 98%, and the representative membrane molecules on lymphocytes such as CD3, CD4, CD8, NK1.1 and NKG2D were detected in the library. These results demonstrated that the cDNA expression library containing transmembrane proteins provided an efficient and fast tool for the study of transmembrane proteins on hepatic lymphocytes.
