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Applied and Environmental Microbiology, August 2008, p. 5139-5145, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00618-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Noncovalent Immobilization of Streptavidin on In Vitro- and In Vivo-Biotinylated Bacterial Magnetic Particles{triangledown}

Yoshiaki Maeda,1 Tomoko Yoshino,1 Masaaki Takahashi,2 Harumi Ginya,2 Junko Asahina,2 Hideji Tajima,2 and Tadashi Matsunaga1*

Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan,1 Precision System Science Co., Ltd., 88 Kamihongo, Matsudo, Chiba 271-0064, Japan2

Received 13 March 2008/ Accepted 9 June 2008

Biotinylated magnetic nanoparticles were constructed by displaying biotin acceptor peptide (BAP) or biotin carboxyl carrier protein (BCCP) on the surface of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1. BAP-displaying BacMPs (BAP-BacMPs) were extracted from bacterial cells and incubated with biotin and Escherichia coli biotin ligase. Then the in vitro biotinylation of BAP-BacMPs was confirmed using alkaline phosphatase-labeled antibiotin antibody. In contrast, BacMPs displaying the intact 149 residues of AMB-1 BCCP (BCCP-BacMPs) and displaying the COOH-terminal 78 residues of BCCP (BCCP78-BacMPs) were biotinylated in AMB-1 cells. The in vivo biotinylation of BCCP-BacMPs and BCCP78-BacMPs was thought to be performed by endogenous AMB-1 biotin ligase. Streptavidin was introduced onto biotinylated BacMPs by simple mixing. In an analysis using tetramethyl rhodamine isocyanate-labeled streptavidin, approximately 15 streptavidin molecules were shown to be immobilized on a single BCCP-BacMP. Furthermore, gold nanoparticle-BacMP composites were constructed via the biotin-streptavidin interaction. The conjugation system developed in this work provides a simple, low-cost method for producing biotin- or streptavidin-labeled magnetic nanoparticles. Various functional materials can be site selectively immobilized on these specially designed BacMPs. By combining the site-selective biotinylation technology and the protein display technology, more innovative and attractive magnetic nanomaterials can be constructed.

* Corresponding author. Mailing address: Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. Phone: 81-42-388-7020. Fax: 81-42-385-7713. E-mail: tmatsuna{at}cc.tuat.ac.jp

{triangledown} Published ahead of print on 20 June 2008.

Applied and Environmental Microbiology, August 2008, p. 5139-5145, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00618-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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