Synthesis of Bacterial Magnetic Particles During Cell Cycle of Magnetospirillum magneticum AMB-1

2001 ◽  
pp. 155-160
Author(s):  
Chen-Dong Yang ◽  
Haruko Takeyama ◽  
Tsuyoshi Tanaka ◽  
Aki Hasegawa ◽  
Tadashi Matsunaga
Keyword(s):  
Cell Cycle ◽  
Magnetic Particles ◽  
Bacterial Magnetic Particles ◽  
Magnetospirillum Magneticum

Synthesis of Bacterial Magnetic Particles During Cell Cycle of Magnetospirillum magneticum AMB-1

2001 ◽  
Vol 91-93 (1-9) ◽  
pp. 155-160 ◽  
Author(s):  
Chen-Dong Yang ◽  
Haruko Takeyama ◽  
Tsuyoshi Tanaka ◽  
Aki Hasegawa ◽  
Tadashi Matsunaga
Keyword(s):  
Cell Cycle ◽  
Magnetic Particles ◽  
Bacterial Magnetic Particles ◽  
Magnetospirillum Magneticum

scholarly journals Assembly of G Protein-Coupled Receptors onto Nanosized Bacterial Magnetic Particles Using Mms16 as an Anchor Molecule

2004 ◽  
Vol 70 (5) ◽  
pp. 2880-2885 ◽  
Author(s):  
Tomoko Yoshino ◽  
Masayoshi Takahashi ◽  
Haruko Takeyama ◽  
Yoshiko Okamura ◽  
Fukuichi Kato ◽  
...  
Keyword(s):  
G Protein ◽  
Magnetic Particles ◽  
Lipid Membrane ◽  
Specific Protein ◽  
G Protein Coupled Receptors ◽  
Native Conformation ◽  
Wide Range ◽  
Bacterial Magnetic Particles ◽  
Magnetospirillum Magneticum ◽  
G Protein Coupled

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.

scholarly journals Inducible Expression of Transmembrane Proteins on Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1

2009 ◽  
Vol 76 (4) ◽  
pp. 1152-1157 ◽  
Author(s):  
Tomoko Yoshino ◽  
Akiko Shimojo ◽  
Yoshiaki Maeda ◽  
Tadashi Matsunaga
Keyword(s):  
Magnetic Particles ◽  
Fluorescent Protein ◽  
Expression System ◽  
Transmembrane Proteins ◽  
Inducible Expression ◽  
Bacterial Cells ◽  
Virus Envelope ◽  
Inducible Expression System ◽  
Bacterial Magnetic Particles ◽  
Magnetospirillum Magneticum

ABSTRACT Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1 are used for a variety of biomedical applications. In particular, the lipid bilayer surrounding BacMPs has been reported to be amenable to the insertion of recombinant transmembrane proteins; however, the display of transmembrane proteins in BacMP membranes remains a technical challenge due to the cytotoxic effects of the proteins when they are overexpressed in bacterial cells. In this study, a tetracycline-inducible expression system was developed to display transmembrane proteins on BacMPs. The expression and localization of the target proteins were confirmed using luciferase and green fluorescent protein as reporter proteins. Gene expression was suppressed in the absence of anhydrotetracycline, and the level of protein expression could be controlled by modulating the concentration of the inducer molecule. This system was implemented to obtain the expression of the tetraspanin CD81. The truncated form of CD81 including the ligand binding site was successfully displayed at the surface of BacMPs by using Mms13 as an anchor protein and was shown to bind the hepatitis C virus envelope protein E2. These results suggest that the tetracycline-inducible expression system described here will be a useful tool for the expression and display of transmembrane proteins in the membranes of BacMPs.

scholarly journals Efficient and Stable Display of Functional Proteins on Bacterial Magnetic Particles Using Mms13 as a Novel Anchor Molecule

2006 ◽  
Vol 72 (1) ◽  
pp. 465-471 ◽  
Author(s):  
Tomoko Yoshino ◽  
Tadashi Matsunaga
Keyword(s):  
Biomedical Applications ◽  
Magnetic Particles ◽  
Protein A ◽  
Bilayer Membrane ◽  
Large Protein ◽  
C Terminus ◽  
Transmission Electron ◽  
Antibody Complex ◽  
Bacterial Magnetic Particles ◽  
Magnetospirillum Magneticum

ABSTRACT Magnetic particles are increasingly used for various biomedical applications because they are easy to handle and separate from biological samples. In this work, a novel anchor molecule was used for targeted protein display onto magnetic nanoparticles. The magnetic bacterium Magnetospirillum magneticum AMB-1 synthesizes intracellular bacterial magnetic particles (BMPs) covered with a lipid bilayer membrane. In our recent research, an integral BMP membrane protein, Mms13, was isolated and used as an anchor molecule to display functional proteins onto BMPs. The anchoring properties of Mms13 were confirmed by luciferase fusion studies. The C terminus of Mms13 was shown to be expressed on the surface of BMPs, and Mms13 was bound to magnetite directly and tightly permitting stable localization of a large protein, luciferase (61 kDa), on BMPs. Consequently, luminescence intensity obtained from BMPs using Mms13 as an anchor molecule was >400 or 1,000 times higher than Mms16 or MagA, which previously were used as anchor molecules. Furthermore, the immunoglobulin G-binding domain of protein A (ZZ) was displayed uniformly on BMPs using Mms13, and antigen was detected by transmission electron microscopy using antibody-labeled gold nanoparticles on a single BMP displaying the ZZ-antibody complex. The results of this study demonstrated the utility of Mms13 as a molecular anchor, which will facilitate the assembly of other functional proteins onto BMPs in the near feature.

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