High-level expression of soluble form of mouse natural killer cell receptor NKR-P1C(B6) in Escherichia coli Daniel Rozbesky ´ a,b , Daniel Kavan b , Josef Chmelík b , Petr Novák a,b , Ondr ˇej Vane ˇk a,b , Karel Bezouška a,b,⇑ a Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-12840 Prague 2, Czech Republic b Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Víden ˇ ská 1083, CZ-14220 Prague 4, Czech Republic article info Article history: Received 26 December 2010 and in revised form 25 January 2011 Available online 1 February 2011 Keywords: Natural killer cell NKR-P1C receptor NK1.1 antigen C-type lectin domain Refolding 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 mono- meric 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 crystal- lization and detailed structural investigations, and is a prerequisite for its use as a probe in ligand iden- tification and antibody epitope mapping experiments. Ó 2011 Elsevier Inc. All rights reserved. Introduction Natural killer (NK) 1 cells represent a subpopulation of large gran- ular lymphocytes that play a key role in elimination of virally in- fected and tumor cells [1]. The cytolytic activity of NK cells depends on sophisticated repertoire of activating and inhibitory receptors which are expressed on their surface. NK receptors can be divided into two families by structural homology: the immuno- globulin-like NK receptors (such as KIRs, LILRs and PIRs) and C-type lectin-like NK receptors (such as Ly49s, CD94/NKG2 and NKR-P1) [2,3]. The latter group of receptors appears to be an important com- ponent of an alternative missing self-system of NK cells based not on the recognition of MHC class I, but on unusual, apparently carbohy- drate-independent lectin–lectin interactions between individual NKR-P1 isoforms and a newly identified family of C-type lectin re- lated (Clr) ligands [4,5]. This receptor system appears to be impor- tant for recognition of tumor cells, viral immunity, and immune recognition of cells subjected to genotoxic stress upon administra- tion of chemotherapeutic compounds in rodents [5–7]. In mice, a to- tal of 7 NKR-P1 receptors and 5 Clr ligands have been identified, and shown to be important for both inhibition and activation of certain lymphocyte subsets. In rats, 5 NKR-P1 receptors and 7 Clr ligands have been identified, although the molecular details of interactions and functions of these individual receptor–ligand pairs are mostly still missing [8,9]. The mouse NKR-P1C (mNKR-P1C) belongs to the disulfide- linked homodimeric type II transmembrane C-type lectin-like receptors encoded by the NKR-P1 gene family. This receptor is composed of C-type lectin-like domain connected by a stalk to the transmembrane and cytoplasmic domain [10,11]. The Nkrp1 (also known as Klrb1) gene cluster is located in the NK gene com- plex (NKC) on chromosome 6 in mice, chromosome 4 in rats, and chromosome 12 in humans [11–13]. The mNKR-P1C receptor cor- responds to the NK1.1 alloantigen, and represents one of the most important surface marker of mouse NK cells and NKT cells in C57BL/6 mice detected by monoclonal antibody PK136 [11]. Although mNKR-P1C had been originally cloned as the NK1.1 anti- gen, analysis revealed that NK1.1 antigen epitope is shared by two receptors, mNKR-P1C and mNKR-P1B [14,15]. The presence of a charged arginine residue in transmembrane segment of mNKR- P1C and absence of cytoplasmatic ITIM motif indicate that 1046-5928/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2011.01.013 ⇑ Corresponding author at: Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-12840 Prague 2, Czech Republic. E-mail address: bezouska@biomed.cas.cz (K. Bezouška). 1 Abbreviations used: CTLD, C-type lectin-like domain; FT-ICR, Fourier transform ion cyclotron resonance; HSQC, heteronuclear single quantum coherence; IPTG, isopro- pyl-b-D-thiogalactopyranoside; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; NK, natural killer; PMSF, phenylmethylsulfonylfluoride; PVDF, polyvinylidenedifluoride; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TFA, trifluoracetic acid; TOF, time of flight. Protein Expression and Purification 77 (2011) 178–184 Contents lists available at ScienceDirect Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep