Bioengineering of bacterial magnetic particles and its application to estrogen receptor-ligand binding assay
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Bioengineering of bacterial magnetic particles and its application to estrogen receptorligand binding assay Tomoko Yoshino, Chihiro Kaji, and Tadashi Matsunaga Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Nakacho, Koganei, 184-8588, Japan ABSTRACT Magnetic 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 magneticum AMB-1. A receptor assay for endocrine-disrupting chemicals using ERLBDdisplaying 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. ERLBDBacMP 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. INTRODUCTION Magnetic particles have been used in various biomedical and environmental applications. The major advantage of magnetic particles is the ease of handling that allows for separation of target molecules from reaction mixtures. Furthermore, these manipulations are comparatively easy to automate. Magnetic particles have been increasingly used as supports for cell separation [1, 2], detection of antigens [3, 4], and removal of heavy metals [5]. Functional groups, oligonucleotides, and proteins have all been assembled previously onto magnetic particles and used as recognition materials for bio-molecular detection assays[6-8]. Bacterial magnetic particles (BacMPs) that are synthesized by magnetotactic bacteria are nano-sized (50–100 nm) and covered by a lipid bilayer membrane[9, 10]. Protein display on BacMPs has been carried out by fusion techniques that involve anchor proteins isolated from the BacMP membrane of Magnetospirillum magneticum AMB-1 [11, 12]. Such functional magnetic nanoparticles have been used for both medical and environmental applications [2, 3, 13]. Various proteins have been identified from BacMP membranes [14-16] and several proteins have been used as anchor proteins for BacMP bioengineering. The transmembrane protein, MagA (46.8 kDa), which was isolated following transposon mutagenesis, was used as an anchor protein, and both luciferase and protein A were displayed on BacMPs [3, 11]. Furthermore, novel anchor proteins Mms16 (16.2 kDa) and Mms13 (12.4 kDa), which were directly isolated from BacMP membranes, were used for efficient protein display on BacMPs [17].
Here, estrogen receptor (ER) was displayed on BacMPs using either Mms16 or Mms13 as the anchor protein for lig
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