Počet záznamů: 1  

Preparation of soluble isotopically labeled NKp30, a human natural cytotoxicity receptor, for structural studies using NMR

  1. 1.
    0389064 - MBÚ 2013 RIV US eng J - Článek v odborném periodiku
    Grave, L. - Tůmová, L. - Mrázek, Hynek - Kavan, Daniel - Chmelík, Josef - Vaněk, Ondřej - Novák, Petr - Bezouška, K.
    Preparation of soluble isotopically labeled NKp30, a human natural cytotoxicity receptor, for structural studies using NMR.
    Protein Expression and Purification. Roč. 86, č. 2 (2012), s. 142-150. ISSN 1046-5928. E-ISSN 1096-0279
    Grant CEP: GA ČR GA303/09/0477; GA ČR GD305/09/H008
    Institucionální podpora: RVO:61388971
    Klíčová slova: NKp30 * NK cell receptor * Uniformly labeled proteins
    Kód oboru RIV: CE - Biochemie
    Impakt faktor: 1.429, rok: 2012

    Using a codon-optimized gene fragment, we report remarkable yields for extracellular domain of human NK cell receptor (NKp30ex) when produced on M9 minimal medium, even with low (2 g/L) glucose concentration. The yields were identical using media containing (NH4Cl)-N-15 or (NH4Cl)-N-15 in combination with all-C-13-D-glucose allowing to produce homogenous soluble monomeric NKp30 in several formats needed for advanced NMR studies. Our optimized protocol now allows to produce routinely 10 mg batches of these NKp30ex proteins per 1 L of M9 production medium in four working days. The purity and identity of the produced proteins were checked by SDS-PAGE, MALDI MS peptide mapping, and high resolution ion cyclotron resonance MS. Analytical ultracentrifugation confirmed the monomeric status of the produced proteins. Long-term stability of the produced protein proved to be very good allowing its use for NMR studies using elevated temperatures. These studies should reveal further details of the interaction of NKp30 with several of its ligands including target cell surface proteins and heparin-derived oligosaccharides. (C) 2012 Elsevier Inc. All rights reserved
    Trvalý link: http://hdl.handle.net/11104/0217995

     
     
Počet záznamů: 1  

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