title: Recombinant expression, in vitro refolding and characterizing disulfide bonds of a mouse inhibitory C-type lectin-like receptor Nkrp1b.
abstract: As a part of the innate immunity, NK (Natural Killer) cells provide an early immune response to different stimuli, e.g. viral infections and tumor growths. However, their functions are more complex; they play an important role in reproduction, alloimmunity, autoimmunity and allergic diseases. NK cell activities require an intricate system of regulation that is ensured by many different receptors on a cell surface which integrate signals from interacting cells and soluble factors. One way to understand NK cell biology is through the structure of NK receptors, which can reveal ligand binding conditions. We present a modified protocol for recombinant expression in Escherichia coli and in vitro refolding of the ligand-binding domain of the inhibitory Nkrp1b (SJL/J) protein. Nkrp1b identity and folding was confirmed using mass spectrometry (accurate mass of the intact protein and evaluation of disulfide bonds) and one-dimensional nuclear magnetic resonance spectroscopy. The intention is to provide the basis for conducting structural studies of the inhibitory Nkrp1b protein, since only the activating Nkrp1a receptor structure is known.
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. lucie.hernychova@biomed.cas.cz or pnovak@biomed.cas.cz.
authors.forename: L
authors.initials: L
authors.lastname: Hernychová
authors.forename: H
authors.initials: H
authors.lastname: Mrázek
authors.forename: L
authors.initials: L
authors.lastname: Ivanova
authors.forename: Z
authors.initials: Z
authors.lastname: Kukačka
authors.forename: J
authors.initials: J
authors.lastname: Chmelík
authors.forename: P
authors.initials: P
authors.lastname: Novák
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D002479:Inclusion Bodies; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D055651:NK Cell Lectin-Like Receptor Subfamily B / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D004220:Disulfides; C527212:Klrb1b protein, mouse; D055651:NK Cell Lectin-Like Receptor Subfamily B; D011994:Recombinant Proteins