- Max Planck Institute of Biophysics
Xia, Hongyan; Liu, Lihong; Reinhart, Christoph; Michel, Hartmut
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 (DIVISO). 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 K(d) value of [12511 NmU-23 (rat) similar to that from transiently transfected COS-7 cells, where hNmU-R1 was first identified. With the aid of 1% n-cloclecyl-beta-D-maltoside (LM)/0.25% cholesteryl hermsuccinate (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 [ 12511 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. (C) 2008 Elsevier B.V. All rights reserved.
GPCR; Membrane protein; Expression; Purification; hNmU-R1
BBA - Biomembranes
2008, Volume: 1778, number: 10, pages: 2203-2209
Publisher: ELSEVIER SCIENCE BV
Biochemistry and Molecular Biology