Site-selective immobilization of streptavidin on enzymatically biotinylated bacterial magnetic particles
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1094-DD07-19
Site-selective immobilization of streptavidin on enzymatically biotinylated bacterial magnetic particles Yoshiaki Maeda1, Tomoko Yoshino1, Haruko Takeyama1, Masaaki Takahashi2, Harumi Ginya2, Junko Asahina2, Hideji Tajima2, and Tadashi Matsunaga1 1
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, 184-8588, Japan 2
Precision System Science Co., Ltd., Japan, 88 Kamihongo, Matsudo, Chiba, 271-0064, Japan
ABSTRACT Biotinylated magnetic nanoparticles were constructed by displaying biotin acceptor peptide (BAP) on the surfaces of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1. Both BAP and green fluorescent protein (GFP) were fused to Mms13 that was isolated from BacMP membranes. The localization of the fusion protein, BAP-Mms13-GFP, was confirmed by fluorescence analysis. BacMPs that expressed BAP-Mms13-GFP (BAP/GFP-BacMPs) were extracted from bacterial cells and incubated with biotin and Escherichia coli biotin ligase. The in vitro biotinylation of BAP/GFP-BacMPs was confirmed using alkaline phosphatase (ALP)-labeled streptavidin. The conjugation system developed in this study provides a method for producing biotin- or streptavidin-labeled magnetic nanoparticles without the use of a crosslinker reagent. Various functional materials can be immobilized site-selectively onto these uniquely designed BacMPs. By combining this site-selective biotinylation technology and protein display methodology, increasingly innovative and attractive magnetic nano-materials can be constructed.
INTRODUCTION Magnetic particles are widely used in various biomedical and environmental applications. A major advantage of magnetic particles is the ease of handling which allows for simple separation of target molecules from reaction mixtures. Among the various magnetic particle composites developed to date, streptavidin-immobilized magnetic particles have shown great potential due to the stability of the biotin-streptavidin interaction and the resulting ability to separate target molecules specifically and efficiently. Magnetospirillum magneticum AMB-1 synthesizes intracellular nano-sized BacMPs (50- to 100-nm) that are surrounded with a lipid bilayer membrane, possess a single magnetic domain of magnetite, and exhibit strong ferrimagnetisms. The display of foreign proteins on BacMPs has been achieved by the expression of a fusion gene combining the target gene and a gene for an anchor protein such as Mms13 that was isolated from the BacMP membrane[1].
Previously, we attempted to immobilize streptavidin onto BacMPs using crosslinker reagents[2-4]. In this method, the crosslinker Sulfo-NHS-LC-LC-biotin, which reacts with the amine group, was initially bound to phosphatidylethanolamine in the BacMP membranes. Following the biotin-modification of BacMPs, streptavidin was successfully immobilized. However, when functional protein-displaying BacMPs were biotinylated, the excess crosslinker reagents also bound to these proteins indiscrimina
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