Novel method for evaluation of chemicals based on ligand-dependent recruitment of GFP labeled coactivator to estrogen receptor displayed on bacterial magnetic particles

AbstractMagnetic particles are used for various biomedical applications because they are easy to both handle and separate from biological samples. Nano-sized bacterial magnetic particles (BacMPs) that display the human estrogen receptor ligand binding domain (ERLBD) on their surfaces were successfully produced by the magnetotactic bacterium,Magnetospirillum magneticumAMB-1. A receptor assay for endocrine-disrupting chemicals using ERLBD-displaying BacMPs was developed. A BacMP membrane-specific protein, Mms16 or Mms13, was used as an anchor protein to localize the ERLBD on the surfaces of BacMPs. ERLBD-BacMP complexes were assayed for competitive binding of alkaline phosphatase-conjugated 17β-estradiol (ALP-E2). Inhibition curve of ALP-E2 to the powerful antagonist, tamoxifen was generated by measuring decreases in luminescence intensity that resulted from the enzymatic reaction of alkaline phosphatase. The overall simplicity of this receptor-binding assay results in a method that can be easily adapted to a high-throughput format.

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Development and evaluation of an automated workstation for single nucleotide polymorphism discrimination using bacterial magnetic particles

Biosensors and Bioelectronics ◽

10.1016/s0956-5663(03)00189-1 ◽

2003 ◽

Vol 19 (4) ◽

pp. 325-330 ◽

Cited By ~ 25

Author(s):

Tsuyoshi Tanaka ◽

Kohei Maruyama ◽

Kiyoushi Yoda ◽

Etsuo Nemoto ◽

Yuuji Udagawa ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Magnetic Particles ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Bacterial Magnetic Particles

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

Applied and Environmental Microbiology ◽

10.1128/aem.70.5.2880-2885.2004 ◽

2004 ◽

Vol 70 (5) ◽

pp. 2880-2885 ◽

Cited By ~ 49

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.

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Molecular Bioengineering of Bacterial Magnetic Particles for Biotechnological Applications

Microbiology Monographs - Magnetoreception and Magnetosomes in Bacteria ◽

10.1007/7171_045 ◽

2006 ◽

pp. 227-254 ◽

Cited By ~ 14

Author(s):

Tadashi Matsunaga ◽

Atsushi Arakaki

Keyword(s):

Magnetic Particles ◽

Biotechnological Applications ◽

Bacterial Magnetic Particles

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Novel Method for Selection of Antimicrobial Peptides from a Phage Display Library by Use of Bacterial Magnetic Particles

Applied and Environmental Microbiology ◽

10.1128/aem.00162-08 ◽

2008 ◽

Vol 74 (24) ◽

pp. 7600-7606 ◽

Cited By ~ 14

Author(s):

Tsuyoshi Tanaka ◽

Yoriko Kokuryu ◽

Tadashi Matsunaga

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Peptides ◽

Phage Display ◽

Magnetic Particles ◽

Peptide Library ◽

Inner Membrane ◽

Phage Display Peptide Library ◽

Wide Range ◽

Bacterial Magnetic Particles ◽

Selection Of

ABSTRACT Antimicrobial peptides were isolated from a phage display peptide library using bacterial magnetic particles (BacMPs) as a solid support. The BacMPs obtained from “Magnetospirillum magneticum” strain AMB-1 consist of pure magnetite (50 to 100 nm in size) and are covered with a lipid bilayer membrane derived from the invagination of the inner membrane. BacMPs are easily purified from a culture of magnetotactic bacteria by magnetic separation. Approximately 4 × 1010 PFU of the library phage (complexity, 2.7 × 109) was reacted with BacMPs. The elution of bound phages from BacMPs was performed by disrupting its membrane with phospholipase D treatment. Six candidate peptides, which were highly cationic and could bind onto the BacMP membrane, were obtained. They exhibited antimicrobial activity against Bacillus subtilis but not against Escherichia coli and Saccharomyces cerevisiae. The amino acid substitution of the selected peptide, KPQQHNRPLRHK (peptide 6-7), to enhance the hydrophobicity resulted in obvious antimicrobial activity against all test microorganisms. The present study shows for the first time that a magnetic selection of antimicrobial peptides from the phage display peptide library was successfully achieved by targeting the actual bacterial inner membrane. This BacMP-based method could be a promising approach for a high-throughput screening of antimicrobial peptides targeting a wide range of species.

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