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Statements

Subject Item
n2:RIV%2F00216208%3A11310%2F12%3A10126855%21RIV13-GA0-11310___
rdf:type
n7:Vysledek skos:Concept
rdfs:seeAlso
http://dx.doi.org/10.1016/j.pep.2012.09.016
dcterms: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.
dcterms: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
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
skos:notation
RIV/00216208:11310/12:10126855!RIV13-GA0-11310___
n7:predkladatel
n17:orjk%3A11310
n3:aktivita
n8:S n8:P n8:I
n3:aktivity
I, P(GA303/09/0477), P(GD305/09/H008), S
n3:cisloPeriodika
2
n3:dodaniDat
n13:2013
n3:domaciTvurceVysledku
n11:5734347 n11:8710740 n11:8732124 n11:8652406 n11:2100592 n11:2491842
n3:druhVysledku
n14:J
n3:duvernostUdaju
n21:S
n3:entitaPredkladatele
n9:predkladatel
n3:idSjednocenehoVysledku
161364
n3:idVysledku
RIV/00216208:11310/12:10126855
n3:jazykVysledku
n18:eng
n3:klicovaSlova
advanced NMR techniques; structural studies; uniformly labeled proteins; NK cell receptor; NKp30
n3:klicoveSlovo
n16:structural%20studies n16:NKp30 n16:NK%20cell%20receptor n16:advanced%20NMR%20techniques n16:uniformly%20labeled%20proteins
n3:kodStatuVydavatele
US - Spojené státy americké
n3:kontrolniKodProRIV
[252B688149D5]
n3:nazevZdroje
Protein Expression and Purification
n3:obor
n15:CE
n3:pocetDomacichTvurcuVysledku
6
n3:pocetTvurcuVysledku
8
n3:projekt
n12:GD305%2F09%2FH008 n12:GA303%2F09%2F0477
n3:rokUplatneniVysledku
n13:2012
n3:svazekPeriodika
86
n3:tvurceVysledku
Vaněk, Ondřej Grave, Lena Bezouška, Karel Mrázek, Hynek Kavan, Daniel Chmelík, Josef Novák, Petr Tůmová, Lucie
n3:wos
000311476000008
s:issn
1046-5928
s:numberOfPages
9
n19:doi
10.1016/j.pep.2012.09.016
n10:organizacniJednotka
11310