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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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 Creators:
Xia, Hongyan1, Author           
Liu, Lihong2, Author
Reinhart, Christoph1, Author           
Michel, Hartmut1, Author                 
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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Free keywords: GPCR; Membrane protein; Expression; Purification; hNmU-R1
 Abstract: 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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Language(s): eng - English
 Dates: 2008-05-082008-01-082008-05-282008-06-132008-10-01
 Publication Status: Issued
 Pages: 7
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1016/j.bbamem.2008.05.017
 Degree: -

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Title: Biochimica et Biophysica Acta-Biomembranes
Source Genre: Journal
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Publ. Info: Amsterdam : Elsevier
Pages: - Volume / Issue: 1778 (10) Sequence Number: - Start / End Page: 2203 - 2209 Identifier: ISSN: 0005-2736
CoNE: https://pure.mpg.de/cone/journals/resource/954926938702