Heterologous expression of human Neuromedin U receptor 1 and its subsequent solubilization and purification.
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-beta-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.
Xia
Hongyan
H
Max-Planck-Institute of Biophysics, 60438 Frankfurt am Main, Germany. Hongyan.Xia@imbim.uu.se
Liu
Lihong
L
Reinhart
Christoph
C
Michel
Hartmut
H
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D051381:Rats; D017981:Receptors, Neurotransmitter / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics / Q000378:metabolism
D017981:Receptors, Neurotransmitter; D011994:Recombinant Proteins; C082444:neuromedin U receptor