Preparation of soluble isotopically labeled NKp30, a human natural cytotoxicity receptor, for structural studies using NMR.
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 (2g/L) glucose concentration. The yields were identical using media containing (15)NH(4)Cl or (15)NH(4)Cl in combination with all-(13)C-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 10mg batches of these NKp30ex proteins per 1L 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.
Department of Biochemistry, Charles University Prague, Praha, Czech Republic.
Lena
L
Grave
Lucie
L
Tůmová
Hynek
H
Mrázek
Daniel
D
Kavan
Josef
J
Chmelík
Ondřej
O
Vaněk
Petr
P
Novák
Karel
K
Bezouška
D000595:Amino Acid Sequence; D000643:Ammonium Chloride / Q000737:chemistry; D001483:Base Sequence; D019149:Bioreactors; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D055628:Natural Cytotoxicity Triggering Receptor 3 / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014461:Ultracentrifugation
D003062:Codon; C431710:NCR3 protein, human; D055628:Natural Cytotoxicity Triggering Receptor 3; D011994:Recombinant Proteins; D000643:Ammonium Chloride