Biomedical Engineering – From Theory to Applications

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396 Biomedical Engineering – From Theory to Applications Strong promoters and stable anchor proteins allowed efficient display of functional proteins on BacMPs. However, the display of particular proteins remained a technical challenge due to the cytotoxic effects of the proteins when they were overexpressed in bacterial cells. Specifically, transmembrane proteins such as G-protein coupled receptors were still difficult to express in magnetotactic bacteria. An inducible protein expression system is often used to control the expression dose and timing of transmembrane proteins. Recently, we developed a tetracycline-inducible protein expression system in M. magneticum AMB-1 to prevent the toxic effects of transmembrane protein expression (Yoshino et al., 2010). This system was implemented to obtain the expression of tetraspanin CD81, where the truncated form of CD81, including the ligand binding site, was successfully expressed on the surface of BacMPs using Mms13 as an anchor protein and the tetracycline-inducible protein expression system. These results suggest that the inducible expression system will be a useful tool for the expression and display of transmembrane and other potentially cytotoxic proteins on the membranes of BacMPs. Currently, many types of functional proteins can be displayed at high levels on magnetic particles due to the modifications described above. Generally, immobilization of proteins onto magnetic particles is performed by chemical cross-linking; however, this can hinder the activity of some proteins. Because the amine-reactive cross-linker can bind to proteins in a random manner, the target proteins may become inactivated. Furthermore, protein orientation on the solid phase is difficult to control during chemical conjugation. To overcome these difficulties, protein display on magnetic particles produced by magnetotactic bacteria through gene fusion is a promising approach, and the techniques have expanded the number of applications. 3. Applications of magnetic particles Magnetic iron oxide particles, such as magnetite (Fe3O4) and maghemite (γ-Fe2O3), are widely used in medical and diagnostic applications such as magnetic resonance imaging (Gleich and Weizenecker, 2005), cell separation (Miltenyi et al., 1990) , drug delivery (Plank et al., 2003), and hyperthermia (Pardoe et al., 2003). To use these particles for biotechnological applications, the surface modification of the magnetic particle with functional molecules such as proteins, peptides, or DNA must be considered. Previously, only DNA- or antibody-immobilized magnetic particles were marketed and used in biotechnology; it was suggested that the techniques for the immobilization of enzymes or receptors were more complicated and time consuming. However, as the methods for assembling functional proteins onto magnetic particles have become simpler and more efficient, the applications of magnetic particles have expanded. Here, the applications of BacMPs displaying functional proteins such as antibody, enzyme, or receptor are described. 3.1 Applications of antibody-magnetic particles Magnetic particles have been widely used as carriers of antibodies for immunoassay, cell separation, and tissue typing (Herr et al., 2006; Tiwari et al., 2003; Weissleder et al., 2005). The use of magnetic particles is advantageous for full automation, minimizing manual labor and providing more precise results (Sawakami-Kobayashi et al., 2003; Tanaka and Matsunaga, 2000). In particular, immunomagnetic particles have been used preferentially in target cell separation from leukocytes (Stampfli et al., 1994; Schratzberger et al., 1997) for in vitro diagnosis, as this provides a more rapid and simple methodology compared with cell sorting using a flow cytometer.
The Potential of Genetically Engineered Magnetic Particles in Biomedical Applications To immobilize antibody, protein A, which is the antibody-binding protein derived from Staphylococcus aureus (Deisenhofer, 1981), has been immobilized on magnetic particles using the sequence of the Z-domain, a synthetic analogue of the IgG-binding B-domain. Staphylococcus protein A consists of a cell wall binding region and five domains, termed C, B, A, D, and E, with C next to the cell wall. The molecular interaction of protein A with IgG is well understood, and the binding sites on the Fc domain of IgG1, -2, and -4 have been characterized. X-ray analysis has revealed that the B-domain of protein A has two contact sites that interact with the Fc domain of IgG (Eliasson and Kogelschatz, 1988) . Based on this knowledge, a synthetic Z-domain, which consists of 58 amino acids and is capable of binding the Fc domain, has been constructed (Lowenadler et al., 1987) . IgG can bind the Zdomain on magnetic particles with uniform orientation. Z-domain was displayed on bacterial magnetic particles using gene fusion techniques and was used to detect human insulin from whole blood by sandwich enzyme immunoassays. The experimental procedure was fully automated using a pipetting robot bearing a magnet (Tanaka and Matsunaga, 2000). Antibody-conjugated BacMPs also can be utilized for cell separation. In general, nano-sized magnetic particles, rather than micro-sized particles, are preferred for cell separation because separated cells with nano-sized magnetic particles on their surfaces can be used in subsequent flow cytometric analysis (Graepler et al., 1998). Additionally, micro-sized magnetic particles are more likely to have inhibitory effects on cell growth and differentiation after magnetic separation. Magnetic separation permits target cells to be isolated directly from crude samples such as blood, bone marrow, tissue homogenates, or cultivation media. Compared to other more conventional methods of cell separation, magnetic separation may be considered a sample enrichment step for further chromatographic and electromigratory analysis. To enrich for target cells, cell surface antigens, such as cluster of differentiation (CD) antigens, were used as markers. CD8, CD14, CD19, CD20, and CD34 positive cells were efficiently enriched from peripheral blood (Kuhara et al., 2004; Matsunaga et al., 2006). The separated CD34 positive cells retained the capability of forming colonies as hematopoietic stem cells. (A) plasma mononuclear cells Peripheral blood HISTOPAQUE Density gradient HISTOPAQUE red blood cells Magnetic Target cells centrifugation separation Direct separation Fig. 4. Schematic illustration of cell separation procedures. (A) The initial separation of peripheral blood mononuclear cells (PBMCs) from whole blood and the subsequent magnetic separation of target cells from PBMCs using magnetic particles followed the common procedure. (B) Target cells were separated directly from whole blood using magnetic particles in the procedure for direct magnetic cell separation. (B) 397
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BIOMEDICAL ENGINEERING - FROM THEOR
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VI Contents Chapter 9 Targeted Magn
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X Preface stand-alone and readers c
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108 Method (Detection) CIEF-tITP-CZ
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110 Method (Detection) Analyte Matr
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120 6. Conclusion Biomedical Engine
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150 5. Conclusions Biomedical Engin
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162 1 st phase : half year 4 2 nd p
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166 7. Innovative active training B
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172 12. Maturing projects’ story
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180 16. Acknowledgments Biomedical
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190 R* M M M M MR*M O O O O M O R*
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196 Fig. 9. Chemical structure of 5
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198 Relative Intensity (AU) Biomedi
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204 2. Preparation of IO nanopartic
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256 3. Deposition techniques Biomed
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454 In this case, the eigenvalues a
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456 where Biomedical Engineering -
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472 Nb b b l l1 Biomedical Engineer
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