Working at the border between innate and adaptive immunity, natural killer (NK) cells play a key role in the immune system by protecting healthy cells and by eliminating malignantly transformed, stressed or virally infected cells. NK cell recognition of a target cell is mediated by a receptor "zipper" consisting of various activating and inhibitory receptors, including C-type lectin-like receptors. Among this major group of receptors, two of the largest rodent receptor families are the NKR-P1 and the Clr receptor families. Although these families have been shown to encode receptor-ligand pairs involved in MHC-independent self-nonself discrimination and are a target for immune evasion by tumour cells and viruses, structural mechanisms of their mutual recognition remain less well characterized. Therefore, we developed a non-viral eukaryotic expression system based on transient transfection of suspension-adapted human embryonic kidney 293 cells to produce soluble native disulphide dimers of NK cell C-type lectin-like receptor ectodomains. The expression system was optimized using green fluorescent protein and secreted alkaline phosphatase, easily quantifiable markers of recombinant protein production. We describe an application of this approach to the recombinant protein production and characterization of native rat NKR-P1B and Clr-11 proteins suitable for further structural and functional studies.

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 12840 Prague Czech Republic

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 12840 Prague Czech Republic EMBL Hamburg c o DESY Building 25A Notkestraße 85 22603 Hamburg Germany

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 12840 Prague Czech Republic Institute of Microbiology The Czech Academy of Sciences Vídeňská 1083 14220 Prague Czech Republic

Department of Immunology University of Toronto 1 King's College Circle M5S 1A8 Toronto ON Canada

Department of Infectious Diseases Robert Koch Institute Seestraße 10 13353 Berlin Germany

Institute of Microbiology The Czech Academy of Sciences Vídeňská 1083 14220 Prague Czech Republic

Institute of Microbiology The Czech Academy of Sciences Vídeňská 1083 14220 Prague Czech Republic Institute of Physics Faculty of Mathematics and Physics Charles University Ke Karlovu 5 12116 Prague Czech Republic

Institute of Physics Faculty of Mathematics and Physics Charles University Ke Karlovu 5 12116 Prague Czech Republic

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$a Vaněk, Ondřej $u Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, 12840, Prague, Czech Republic. ondrej.vanek@natur.cuni.cz.

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$a Production of recombinant soluble dimeric C-type lectin-like receptors of rat natural killer cells / $c O. Vaněk, P. Celadova, O. Skořepa, J. Bláha, B. Kalousková, A. Dvorská, E. Poláchová, H. Pucholtová, D. Kavan, P. Pompach, K. Hofbauerová, V. Kopecký, A. Mesci, S. Voigt, JR. Carlyle,

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$a Working at the border between innate and adaptive immunity, natural killer (NK) cells play a key role in the immune system by protecting healthy cells and by eliminating malignantly transformed, stressed or virally infected cells. NK cell recognition of a target cell is mediated by a receptor "zipper" consisting of various activating and inhibitory receptors, including C-type lectin-like receptors. Among this major group of receptors, two of the largest rodent receptor families are the NKR-P1 and the Clr receptor families. Although these families have been shown to encode receptor-ligand pairs involved in MHC-independent self-nonself discrimination and are a target for immune evasion by tumour cells and viruses, structural mechanisms of their mutual recognition remain less well characterized. Therefore, we developed a non-viral eukaryotic expression system based on transient transfection of suspension-adapted human embryonic kidney 293 cells to produce soluble native disulphide dimers of NK cell C-type lectin-like receptor ectodomains. The expression system was optimized using green fluorescent protein and secreted alkaline phosphatase, easily quantifiable markers of recombinant protein production. We describe an application of this approach to the recombinant protein production and characterization of native rat NKR-P1B and Clr-11 proteins suitable for further structural and functional studies.

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$a Celadova, Petra $u Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, 12840, Prague, Czech Republic.

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$a Skořepa, Ondřej $u Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, 12840, Prague, Czech Republic.

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$a Bláha, Jan $u Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, 12840, Prague, Czech Republic. EMBL Hamburg, c/o DESY, Building 25A, Notkestraße 85, 22603, Hamburg, Germany.

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$a Kalousková, Barbora $u Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, 12840, Prague, Czech Republic.

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$a Poláchová, Edita $u Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, 12840, Prague, Czech Republic.

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$a Kavan, Daniel $u Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, 12840, Prague, Czech Republic. Institute of Microbiology, The Czech Academy of Sciences, Vídeňská 1083, 14220, Prague, Czech Republic.

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$a Hofbauerová, Kateřina $u Institute of Microbiology, The Czech Academy of Sciences, Vídeňská 1083, 14220, Prague, Czech Republic. Institute of Physics, Faculty of Mathematics and Physics, Charles University, Ke Karlovu 5, 12116, Prague, Czech Republic.

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$a Kopecký, Vladimír $u Institute of Physics, Faculty of Mathematics and Physics, Charles University, Ke Karlovu 5, 12116, Prague, Czech Republic.

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$a Mesci, Aruz $u Department of Immunology, University of Toronto, 1 King's College Circle, M5S 1A8, Toronto, ON, Canada.

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$a Voigt, Sebastian $u Department of Infectious Diseases, Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.

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$a Carlyle, James R $u Department of Immunology, University of Toronto, 1 King's College Circle, M5S 1A8, Toronto, ON, Canada.

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