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

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rdf:type	skos:Concept http://linked.opendata.cz/ontology/domain/vavai/Vysledek
rdfs:seeAlso	http://dx.doi.org/10.1016/j.pep.2012.09.016
Description	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. 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. (en)
Title	Preparation of soluble isotopically labeled NKp30, a human natural cytotoxicity receptor, for structural studies using NMR Preparation of soluble isotopically labeled NKp30, a human natural cytotoxicity receptor, for structural studies using NMR (en)
skos:prefLabel	Preparation of soluble isotopically labeled NKp30, a human natural cytotoxicity receptor, for structural studies using NMR Preparation of soluble isotopically labeled NKp30, a human natural cytotoxicity receptor, for structural studies using NMR (en)
skos:notation	RIV/00216208:11310/12:10126855!RIV13-GA0-11310___
http://linked.open...avai/predkladatel	Přírodovědecká fakulta
http://linked.open...avai/riv/aktivita	I P S
http://linked.open...avai/riv/aktivity	I, P(GA303/09/0477), P(GD305/09/H008), S
http://linked.open...iv/cisloPeriodika	2
http://linked.open...vai/riv/dodaniDat	2013
http://linked.open...aciTvurceVysledku	Bezouška, Karel Kavan, Daniel Novák, Petr Vaněk, Ondřej Mrázek, Hynek Tůmová, Lucie
http://linked.open.../riv/druhVysledku	J - Článek v odborném periodiku
http://linked.open...iv/duvernostUdaju	S - Úplné a pravdivé údaje nepodléhající ochraně podle zvláštních právních předpisů
http://linked.open...titaPredkladatele	Univerzita Karlova v Praze / Přírodovědecká fakulta
http://linked.open...dnocenehoVysledku	161364
http://linked.open...ai/riv/idVysledku	RIV/00216208:11310/12:10126855
http://linked.open...riv/jazykVysledku	eng - angličtina
http://linked.open.../riv/klicovaSlova	advanced NMR techniques; structural studies; uniformly labeled proteins; NK cell receptor; NKp30 (en)
http://linked.open.../riv/klicoveSlovo	NK cell receptor NKp30 advanced NMR techniques structural studies uniformly labeled proteins
http://linked.open...odStatuVydavatele	US - Spojené státy americké
http://linked.open...ontrolniKodProRIV	[252B688149D5]
http://linked.open...i/riv/nazevZdroje	Protein Expression and Purification
http://linked.open...in/vavai/riv/obor	CE
http://linked.open...ichTvurcuVysledku	6 (xsd:int)
http://linked.open...cetTvurcuVysledku	8 (xsd:int)
http://linked.open...vavai/riv/projekt	Preparation, biotransformation, and optimalization of compounds with antitumor and antimicrobial effects Glycomic and proteomic analysis of the molecular mechanisms of elimination of clinically relevant tumors by killer cells of the immune system
http://linked.open...UplatneniVysledku	2012
http://linked.open...v/svazekPeriodika	86
http://linked.open...iv/tvurceVysledku	Bezouška, Karel Kavan, Daniel Novák, Petr Tůmová, Lucie Vaněk, Ondřej Chmelík, Josef Mrázek, Hynek Grave, Lena
http://linked.open...ain/vavai/riv/wos	000311476000008
issn	1046-5928
number of pages	9 (xsd:int)
http://bibframe.org/vocab/doi	10.1016/j.pep.2012.09.016
http://localhost/t...ganizacniJednotka	11310
is http://linked.open...avai/riv/vysledek of	Preparation of soluble isotopically labeled NKp30, a human natural cytotoxicity receptor, for structural studies using NMR

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