Affiliation:
1. Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan
Abstract
ABSTRACT
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.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Reference34 articles.
1. Arakaki, A., J. Webb, and T. Matsunaga. 2002. A novel protein tightly bound to bacterial magnetic particles in Magnetospirillum magneticum strain AMB-1. J. Biol. Chem.278:8745-8750.
2. Babcock, G. J., T. Mirzabekov, W. Wojtowicz, and J. Sodroski. 2001. Ligand binding characteristics of CXCR4 incorporated into paramagnetic proteoliposomes. J. Biol. Chem.276:38433-38440.
3. Benson, S. A., M. N. Hall, and T. J. Silhavy. 1985. Genetic analysis of protein export in Escherichia coli K12. Annu. Rev. Biochem.54:101-134.
4. Clackson, T., H. R. Hoogenboom, A. D. Griffiths, and G. Winter. 1991. Making antibody fragments using phage display libraries. Nature352:624-628.
5. Daugherty, P. S., M. J. Olsen, B. L. Iverson, and G. Georgiou. 1999. Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface. Protein Eng.12:613-621.
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