Heterologous expression of human Neuromedin U receptor 1 and its subsequent solubilization and purification |
| |
Authors: | Hongyan Xia Lihong Liu Hartmut Michel |
| |
Affiliation: | a Max-Planck-Institute of Biophysics, 60438 Frankfurt am Main, Germany b Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Box 7036, 75007 Uppsala, Sweden |
| |
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. |
| |
Keywords: | BHK-21, Baby hamster kidney BCIP, 5-Bromo-4-Chloro-3-Indolyl Phosphate p-Toluidinium CHS, Cholesteryl hemisuccinate HEGA-10, Decanoyl-N-hydoxyethylglucamide DMSO, Dimethyl sulfoxide DMEM, Dulbecco's Modified Eagle's Medium E. coli, Escherichia coli FCS, Fetal calf serum FOS-12, FOS-choline-12 FOS-14, FOS-choline-14 FOS-16, FOS-choline-16 GPCR, G protein-coupled receptor hNmU-R1, human Neuromedin U receptor 1 MOI, Multiplicity of Infection LM, n-Dodecyl-β- smallcaps" >d-maltoside NTA, Ni-nitrilotriacetic acid NBT, Nitro-Blue Tetrazolium Chloride OG, Octyl-β- smallcaps" >d-glucopyronoside PMSF, Phenymethylsulfonylfluoride SFV, Semliki Forest Virus Sf9, Spodoptera frugiperda TSA, Trichostatin A |
本文献已被 ScienceDirect 等数据库收录! |
|