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  Heterologous expression of human Neuromedin U receptor 1 and its subsequent solubilization and purification

Xia, H., Liu, L., Reinhart, C., & Michel, H. (2008). Heterologous expression of human Neuromedin U receptor 1 and its subsequent solubilization and purification. Biochimica et Biophysica Acta-Biomembranes, 1778(10), 2203-2209. doi:10.1016/j.bbamem.2008.05.017.

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Xia, Hongyan1, Autor           
Liu, Lihong2, Autor
Reinhart, Christoph1, Autor           
Michel, Hartmut1, Autor                 
Affiliations:
1Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              
2Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Box 7036, 75007 Uppsala, Sweden, ou_persistent22              

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Schlagwörter: GPCR; Membrane protein; Expression; Purification; hNmU-R1
 Zusammenfassung: Human Neuromedin U receptor 1 (hNmU-R1) is a member of G protein-coupled receptor family. For structural determination of hNmU-R1, the production of hNmU-R1 in milligram amounts is a prerequisite. Here we reported two different eukaryotic expression systems, namely, Semliki Forest virus (SFV)/BHK-21 and baculovirus/Spodoptera frugiperda (Sf9) cell systems for overproduction of this receptor. In the SFV-based expression system, hNmU-R1 was produced at a level of 5 pmol receptor/mg membrane protein and the yield could be further increased to 22 pmol receptor/mg membrane protein by supplementation with 2% dimethyl sulfoxide (DMSO). Around 8 pmol receptor/mg membrane protein could be achieved in baculovirus-infected Sf9 cells. The recombinant hNmU-R1 from SFV- and baculovirus-based systems was functional, with a Kd value of [125I] NmU-23 (rat) similar to that from transiently transfected COS-7 cells, where hNmU-R1 was first identified. With the aid of 1% n-dodecyl-β-d-maltoside (LM)/0.25% cholesteryl hemisuccinate (CHS), the yield of functional hNmU-R1 could reach 80%. The recombinant receptor from Sf9 cells was purified to homogeneity. The specific binding of the purified receptor to [125I] NmU-23 (rat) indicated that the receptor is bioactive. This is the first report of successful solubilization and purification of hNmU-R1, and will enable functional and structural studies of the hNmU-R1.

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Sprache(n): eng - English
 Datum: 2008-05-082008-01-082008-05-282008-06-132008-10-01
 Publikationsstatus: Erschienen
 Seiten: 7
 Ort, Verlag, Ausgabe: -
 Inhaltsverzeichnis: -
 Art der Begutachtung: Expertenbegutachtung
 Identifikatoren: DOI: 10.1016/j.bbamem.2008.05.017
 Art des Abschluß: -

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Titel: Biochimica et Biophysica Acta-Biomembranes
Genre der Quelle: Zeitschrift
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Ort, Verlag, Ausgabe: Amsterdam : Elsevier
Seiten: - Band / Heft: 1778 (10) Artikelnummer: - Start- / Endseite: 2203 - 2209 Identifikator: ISSN: 0005-2736
CoNE: https://pure.mpg.de/cone/journals/resource/954926938702