Assembly of G protein-coupled receptors onto nanosized bacterial magnetic particles using Mms16 as an anchor molecule.
G protein-coupled receptors (GPCRs) play a central role in a wide range of biological processes and are prime targets for drug discovery. GPCRs have large hydrophobic domains, and therefore purification of GPCRs from cells is frequently time-consuming and typically results in loss of native conformation. In this work, GPCRs have been successfully assembled into the lipid membrane of nanosized bacterial magnetic particles (BMPs) produced by the magnetic bacterium Magnetospirillum magneticum AMB-1. A BMP-specific protein, Mms16, was used as an anchor molecule, and localization of heterologous Mms16 on BMPs was confirmed by luciferase fusion studies. Stable luminescence was obtained from BMPs bearing Mms16 fused with luciferase at the C-terminal region. D1 dopamine receptor (D1R), a GPCR, was also efficiently assembled onto BMPs by using Mms16 as an anchor molecule. D1R-BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants. After washing, the complexes were ready to use for analysis. This system conveniently refines the native conformation of GPCRs without the need for detergent solubilization, purification, and reconstitution after cell disruption.
Yoshino
Tomoko
T
Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.
Takahashi
Masayoshi
M
Takeyama
Haruko
H
Okamura
Yoshiko
Y
Kato
Fukuichi
F
Matsunaga
Tadashi
T
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008280:Magnetics; D036103:Nanotechnology; D017447:Receptors, Dopamine D1 / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D012244:Rhodospirillaceae / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D005290:Ferric Compounds; D017447:Receptors, Dopamine D1; D043562:Receptors, G-Protein-Coupled; D020558:GTP Phosphohydrolases; C445324:Mms16 protein, Magnetospirillum magneticum