Bacterial Magnetic Particles Research Articles

Rapid enrichment and determination of phosphopeptides using bacterial magnetic particles via both strong and weak interactions

Bacterial magnetic nanoparticles (BMP) are used in rapid enrichment and determination of phosphorylated peptides. Fe(3+) and Zr(4+) can be easily immobilized on the surface of BMP without any other modification, and the metal ion immobilized BMP can enrich phosphopeptides rapidly through strong coordination bonding. It is found that the Zr(4+)-immobilized BMP can enrich the multiphosphorylated peptides while Fe(3+)-immobilized BMP preferred the singly phosphorylated peptides. Fe(3+)-immobilized BMP could detect phosphorylated peptides under lower concentration than that of Zr(4+) owing to the larger amount of Fe(3+) on BMPs. Besides, BMP itself can enrich one kind of phosphorylated peptide from alpha-casein digest directly through weak interactions between BMP and phosphorylated peptides.

Inducible expression of transmembrane proteins on bacterial magnetic particles in Magnetospirillum magneticum AMB-1.

Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1 are used for a variety of biomedical applications. In particular, the lipid bilayer surrounding BacMPs has been reported to be amenable to the insertion of recombinant transmembrane proteins; however, the display of transmembrane proteins in BacMP membranes remains a technical challenge due to the cytotoxic effects of the proteins when they are overexpressed in bacterial cells. In this study, a tetracycline-inducible expression system was developed to display transmembrane proteins on BacMPs. The expression and localization of the target proteins were confirmed using luciferase and green fluorescent protein as reporter proteins. Gene expression was suppressed in the absence of anhydrotetracycline, and the level of protein expression could be controlled by modulating the concentration of the inducer molecule. This system was implemented to obtain the expression of the tetraspanin CD81. The truncated form of CD81 including the ligand binding site was successfully displayed at the surface of BacMPs by using Mms13 as an anchor protein and was shown to bind the hepatitis C virus envelope protein E2. These results suggest that the tetracycline-inducible expression system described here will be a useful tool for the expression and display of transmembrane proteins in the membranes of BacMPs.

Protein Display onto Nano-sized Bacterial Magnetic Particles for Receptor Analysis

Magnetic particles offer vast potential in ushering new techniques, especially in biomedical applications, as they can be easily manipulated by magnetic force. Magnetotactic bacteria synthesize nano-sized biomagnetites, otherwise known as bacterial magnetic particles (BacMPs) that are individually enveloped by a lipid bilayer membrane. BacMPs are ultrafine magnetite crystals (50-100 nm diameters) with uniform morphology produced by Magnetospirillum magneticum AMB-1. Based on our elucidations on the molecular mechanism of BacMP formation in M. magneticum AMB-1, functional nanomaterials have been designed. Through genetic engineering, functional proteins such as enzymes, antibodies, and receptors were successfully displayed onto BacMPs. Here, display techniques of functional proteins onto nano-sized BacMPs and its applications to ligand binding assays were described. Dopamine receptor, which is a member of G protein-coupled receptors, was successfully displayed onto BacMPs. This system makes possible the convenient acquisition of the native conformation of membrane proteins without the need for detergent solubilization, purification and reconstitution after cell disruption. Furthermore, estrogen receptor, which is one of nuclear receptors, was also displayed onto BacMPs. The assay using BacMPs displaying estrogen receptor could discriminate full agonists, partial agonists, or antagonists. The elucidation of the mechanism of BacMP synthesis has provided a roadmap for the design of novel nano-biomaterials that would play a useful role in multidisciplinary fields.

CLONING OF A GENE ENCODING PROTEIN BELONGING TO ABC TRANSPORTER INVOLVED IN BACTERIAL MAGNETIC PARTICLE SYNTHESIS IN MAGNETOSPIRILLUM MAGNETICUM AMB-1

Magnetospirillum magneticum AMB-1 synthesizes intracellular magnetic particles, magnetite (Fe3O4), enveloped by membrane called magnetosome under micro-aerobic conditions. Initial study of random transposon-based mutagenesis generated 62 nonmagnetic mutants of AMB-1 in a mini-Tn5 library. In order to identify a gene involved in bacterial magnetic particle (BMP) synthesis in the magnetic bacterium M. magneticum AMB-1, a nonmagnetic mutant from the library designated as NMA38-4, was analyzed. h e amino acid sequence deduced from the gene directly interrupted by transposon, ORF4 (1482 bp), showed homology to ATP binding cassette (ABC) transporter of Mesorhizobium loti with 62 % identity and 74 % similarity. It was strongly indicated by the occurrence of putative consensus sequence of ATP-binding motifs (ATP-binding protein). h e ORF4 was subsequently cloned in pET-15b and the recombinant ORF4-Histag fusion protein was heterologously expressed in Escherichia coli BL21 (DE3) pLysS. A 55 kDa protein corresponding to the ORF4-Histag fusion protein was obtained after purifi cation using Ni-NTA column. h is is the fi rst report describing a gene cluster containing gene encoding protein belonging to ABC transporter organized in an operon which is involved in BMP synthesis. Key words: Magnetospirillum magneticum AMB-1, Bacterial Magnetic Particle (BMP), ATP Binding Cassette (ABC) Transporter, transposon mutagenesis.

High‐Sensitivity Detection of Fruit Tree Viruses Using Bacterial Magnetic Particles

Prunus necrotic ring spot virus (PNRSV) and grapevine fanleaf virus (GFLV) were detected by fluoroimmunoassay using bacterial magnetic particles (BMPs), and a double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). For the fluoroimmunoassay, fluorescein isothiocyanate labeled anti-PNRSV antibody or anti-GFLV antibody was conjugated onto BMPs of Magnetospirillum gryphiswaldense MSR-1. With this method, a very low minimum antigen concentration (1 x 10(6) dilution of the original sample concentration) could be detected. Using DAS-ELISA, the minimum antigen detection concentration was the original sample concentration. Thus, comparing these two methods, a BMP-based method could increase the sensitivity up to six orders of magnitude (10(6)) higher than an ELISA-based method of detection PNRSV and GFLV.

Nano‐sized bacterial magnetic particles displaying pyruvate phosphate dikinase for pyrosequencing

There is a high demand for inexpensive and high-throughput DNA sequencing technologies in molecular biology and applied biosciences. In this study, novel nano-sized magnetic particles displaying enzymes for pyrosequencing, a rather novel bioluminometric DNA sequencing method based on the sequencing-by-synthesis principle by employing a cascade of several enzymatic reactions, was developed. A highly thermostable enzyme, pyruvate phosphate dikinase (PPDK) which converts PPi to ATP was successfully expressed onto bacterial magnetic particles (BacMPs) using a novel protein display system of Magnetospirillum magneticum AMB-1. The enzymatic stability of BacMPs displaying PPDK (PPDK-BacMPs) to pH and temperature was evaluated and its broad range of properties was shown. Subsequently, PPDK-BacMPs were applied in pyrosequencing and a target oligonucleotide was successfully sequenced. The PPDK enzyme displayed on BacMPs was shown to be recyclable in each sequence reaction as they can be manipulated by magnetic force. It was concluded that nano-sized PPDK-BacMPs are useful for the scale down of pyrosequencing reaction volumes, thus, permitting high-throughput. The recycling of enzymes was also shown to be promising and applicable for the development of an inexpensive DNA sequencing at a low running cost.

Magnetic Separation of Melanoma-Specific Cytotoxic T Lymphocytes from a Vaccinated Melanoma Patient’s Blood Using MHC/Peptide Complex-Conjugated Bacterial Magnetic Particles

Target antigen-specific cytotoxic T lymphocytes (CTLs) play a key role in anticancer and antivirus immunity in the body, and purification of CTLs from heterogeneous immune cells is desired for an efficient cancer immunotherapy and fundamental research. Herein, a novel magnetic nanoparticle conjugated with major histocompatibility complex (MHC)/peptide complexes was developed for the magnetic separation of melanoma-specific CTLs. To conjugate biotinylated MHC/peptide complexes on nanosized bacterial magnetic particles (BacMPs), which were synthesized intracellularly by magnetotactic bacteria, phased modification of biotin and streptavidin onto BacMPs was investigated. When biotin was modified on BacMPs, a polyethylene oxide (PEO)-linker contributed to the maintenance of the high dispersion properties of BacMPs. Furthermore, nonspecific binding of BacMPs to cell surface was prevented by controlling the level of streptavidin bound on BacMPs and through PEO blocking of the empty streptavidin sites on BacMPs. Finally, single-step magnetic separation of melanoma-specific CTLs was demonstrated using developed MHC/MAGE-1 A24 peptide complex-conjugated BacMPs. Melanoma-reactive cells and melanoma-specific CTLs were successfully separated from stimulated peripheral blood mononuclear cells derived from a vaccinated melanoma patient with 93.1% and 87.7% purity, respectively, and specificity of antigen recognition and cytokine secretion from separated CTLs were confirmed. The potential of MHC/peptide complex-conjugated BacMPs was indicated for efficient separation of antigen-specific CTLs in cancer immunotherapy and fundamental research.

Magnetic Separation of Human Podocalyxin-like Protein 1 (hPCLP1)-Positive Cells from Peripheral Blood and Umbilical Cord Blood Using Anti-hPCLP1 Monoclonal Antibody and Protein A Expressed on Bacterial Magnetic Particles

Hemangioblasts are common progenitors of hematopoietic and angiogenic cells, which have been demonstrated in the mouse to possess a unique cell surface marker, podocalyxin-like protein 1 (PCLP1) (Hara, T. et al., Immunity, 11: 567-578. 1999). In this study, we prepared a novel monoclonal antibody against human PCLP1 (hPCLP1) and attempted to isolate human hematopoietic progenitor cells from umbilical cord blood and peripheral blood using nano-sized bacterial magnetic particles (BacMPs) coupled with the anti-hPCLP1 antibody. Flow cytometric analysis demonstrated that the purity of separated hPCLP1-positive cells from peripheral blood was approximately 95% whereas peripheral blood mononuclear cells contained only 0.1% PCLP1+ cells. Umbilical cord blood was demonstrated to be a better source for PCLP1+ cells than peripheral blood. These results suggest that the separation of human PCLP1+ cells using BacMPs with anti-hPCLP1 were extremely effective and may be useful as a means to prepare human hematopoietic progenitor cells.

Novel nanocomposites consisting of in vivo-biotinylated bacterial magnetic particles and quantum dots for magnetic separation and fluorescent labeling of cancer cells

Novel nanocomposites consisting of nano-sized bacterial magnetic particles (BacMPs) and semiconductor quantum dots (QD) were developed for use in targeting and identifying cancer cells. BacMPs are synthesized by magnetotactic bacterium, Magnetospirillum magneticumAMB-1 and can display functional proteins using gene fusion techniques. In this study, two functional proteins, biotin carboxyl carrier protein (BCCP), derived from AMB-1 and protein G, derived from Streptococcus were displayed on BacMPs. BCCP was biotinylated in AMB-1 cells by endogenous biotin ligase. After purification of in vivo-biotinylated BacMPs, streptavidin and antibodies were immobilized on protein G-BCCP-displaying BacMPs (protein G-BCCP-BacMPs) viabiotin and protein G, respectively. Furthermore, multi-color labeling of the protein G-BCCP-BacMPs was achieved with streptavidin-labeled QD. Using streptavidin-QD/protein G-BCCP-BacMP nanocomposites, we successfully demonstrated magnetic manipulation and fluorescent labeling of lung cancer cells.

Direct magnetic separation of immune cells from whole blood using bacterial magnetic particles displaying protein G

Direct separation of target cells from mixed population, such as peripheral blood, umbilical cord blood, and bone marrow, is an essential technique for various therapeutic or diagnosis applications. In this study, novel particles were fabricated, and direct magnetic separation of immune cells from whole blood using such particles was performed. The magnetotactic bacterium Magnetospirillum magneticum AMB-1 synthesizes intracellular bacterial magnetic particles (BacMPs), and protein G was expressed on the surface of the BacMPs by gene fusion techniques with anchor proteins isolated from BacMP membrane. The BacMPs displaying protein G (protein G-BacMPs) had high binding capabilities to a wide range of antibody types, and various versions of protein G-BacMPs binding with different anti-CD monoclonal antibodies were constructed. Consequently, direct magnetic separation of immune cells from whole blood using protein G-BacMPs binding with anti-CD monoclonal antibodies was demonstrated. B lymphocytes (CD19+ cells) or T lymphocytes (CD3+ cells), which represent less than 0.05% in whole blood cells, were successfully separated at a purity level of more than 96%. This level was superior to that from previous reports using other magnetic separation approaches. The results of this study demonstrate the utility of protein G-BacMP and this particle may become a powerful tool for various therapeutic or diagnosis applications.

Novel method for selection of antimicrobial peptides from a phage display library by use of bacterial magnetic particles.

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 x 10(10) PFU of the library phage (complexity, 2.7 x 10(9)) 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.

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

We established a novel method to evaluate endocrine disrupting chemicals (EDCs) by assembling the estrogen receptor–ligand binding domain (ERLBD) and GFP labeled coactivator on magnetic nanoparticles. EDC can promote or inhibit coactivator recruitment to the ligand–ERLBD complex. ERLBD was displayed on the surface of nano-sized bacterial magnetic particles (BacMPs) produced by the magnetic bacterium, Magnetospirillum magneticum AMB-1. Our method resulted in 38 molecules of ERLBD molecules on a BacMPs with diameter of 75 nm. Furthermore, ligand-dependent recruitment assays of GFP labeled coactivator to ERLBD–BacMPs was performed by measuring the fluorescence intensity. 17β-estradiol (E2), estriol, diethylstilbestrol, zeralenone (full agonist), octylphenol (partial agonist) and ICI 182,780 (antagonist) were evaluated by this method. Full agonists tested showed increased fluorescence with increasing agonist concentration. Octylphenol had lower fluorescence intensity than E2. ICI 182,780 did not produce any fluorescence. The method developed in this study can evaluate the estrogenic potential of chemicals by discriminating whether they are an ER full agonist, partial agonist, or antagonist. Finally, this method is amenable adaptation into a high throughput format by using automated magnetic separation.

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

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.

Formation of magnetite by bacteria and its application.

Magnetic particles offer high technological potential since they can be conveniently collected with an external magnetic field. Magnetotactic bacteria synthesize bacterial magnetic particles (BacMPs) with well-controlled size and morphology. BacMPs are individually covered with thin organic membrane, which confers high and even dispersion in aqueous solutions compared with artificial magnetites, making them ideal biotechnological materials. Recent molecular studies including genome sequence, mutagenesis, gene expression and proteome analyses indicated a number of genes and proteins which play important roles for BacMP biomineralization. Some of the genes and proteins identified from these studies have allowed us to express functional proteins efficiently onto BacMPs, through genetic engineering, permitting the preservation of the protein activity, leading to a simple preparation of functional protein-magnetic particle complexes. They were applicable to high-sensitivity immunoassay, drug screening and cell separation. Furthermore, fully automated single nucleotide polymorphism discrimination and DNA recovery systems have been developed to use these functionalized BacMPs. The nano-sized fine magnetic particles offer vast potential in new nano-techniques.

Magnetic cell separation using nano‐sized bacterial magnetic particles with reconstructed magnetosome membrane

Magnetic nanoparticles produced by magnetotactic bacterium, bacterial magnetic particles (BacMPs), covered with a lipid bilayer membrane (magnetosome membrane) can be used to separate specific target cells from heterogeneous mixtures because they are easily manipulated by magnets and it is easy to display functional proteins on their surface via genetic engineering. Despite possessing unique and valuable characteristics, the potential toxicity of BacMPs to the separated cells has not been characterized in detail. Here, a novel technique was developed for the reconstruction of magnetosome membrane of BacMPs expressing protein A (protein A-BacMPs) to reduce cytotoxicity and the newly developed nanomaterial was then used for magnetic cell separation. The development of the magnetosome membrane-reconstructed protein A-BacMP was based on the characteristics of the Mms13 anchor protein, which strongly binds to the magnetite surface of BacMPs. Treatment of protein A-BacMPs with detergents removed contaminating proteins but did not affect retention of Mms13-protein A fusion proteins. The particle surfaces were then reconstructed with phosphatidylcholine. The protein A-BacMPs containing reconstructed magnetosome membranes remained dispersible and retained the ability to immobilize antibody. In addition, they contained few membrane surface proteins and endotoxins, which were observed on non-treated protein A-BacMPs. Magnetic separation of monocytes and B-lymphocytes from the peripheral blood was achieved with high purity using magnetosome membrane-reconstructed protein A-BacMPs.

The Development of Miniaturized Extraction Tool for the Gene Biosensor

Analysis of DNA has represented the deep insight of diagnostic and clinical information in individuals for detecting and treating genetic diseases related to loss of heterozygosity, mutation, gene copy number, and genotype. Traditional DNA assays involve extraction of DNA from whole blood, amplification by the polymerase chain reaction, and subsequent separation using slab gel electrophoresis. Although this process is known to be reasonably successful, it is generally time-consuming and unsuitable for high-throughput analysis. A smaller reaction volume would be beneficial to reduce the cost of reagents and the amount of waste generated. Extraction of DNA from cellular constituents is also a key for most nucleic acid-based drug discovery and clinical diagnostic applications. It is important that inhibitors or interfering substances from the cells and extracellular environment should be removed prior to PCR analysis. Traditionally, the organic-based extraction and ethanol precipitation have been used to isolate and concentrate DNA prior to digestion and amplification. Although this procedure could handle about 10 to 20 μL of blood, it costs time and efforts due to many steps including cells lysis, several unltracentrifuges and precipitations. Commercial kits such as Qiagen DNA Stool Mini Kit and QIAamp DNA kit have provided relatively good extraction efficiency, however, drawbacks are increasing number of steps (more than 20 steps) for blood treatment and large sample volume (more than 250 μL). Several papers have been reported in order to overcome the disadvantages of the extraction of DNA from whole blood. For example, dendrimer-modified bacterial magnetic particles were employed based on the interaction of negatively-charged DNA and cations on dendrimer. This electrostatic interaction was also utilized on amino silane coated-polydimethylsiloxane (PDMS) microchip with the blood volume of less than 20 μL. Glass microchip with heat-treated frit was used for the extraction and detection of DNA from spores of the vaccine strain. Although somewhat effective extraction was achieved with magnetic particles or microchips, many shortcomings such as long modification time, intricate control of coating, and high cost for microchip production should be considered before the experiment. Recently, DNA purification process with adsorption of DNA onto a solid surface via hydrogen boding to silica and via electrostatic interactions has been developed. This approach allows inhibiting species to be removed before the purified DNA is eluted from the surface and affords the opportunity for miniaturization. In this paper, a capillary-based DNA extraction tool was developed for the blood volume of less than 3 μL. Only 3 steps were required before PCR amplification of extracted DNA. Glutathione-S-transferase genes related to bladder and breast cancer was tested with our extraction tool. The conditions for frit formation by photopolymerization including the ratios of photoinitiators, the frit length, UV exposure time, and the size of the silica bead particles were examined and optimized. Figure 1 shows our miniaturized extraction tool using fused-silica capillary tube (520 μm i.d.). As shown in Figure 1(a) and (b), the tool was built with two parts; the region for silica particles and the frit for packing of the particles. Packed silica particles provide the isolation and extraction of genomic DNA while the frit holds the silica particles. Compared to the sleeve type packing with glass fiber, our tool is simpler in construction, and easier to fill the particles in the capillary tube. For the construction of the frit, the choice of the photoinitiator is important. Bis acyl phosphine or α-hydroxyketone is a well-known chemical class that absorbs UV energy from the light source and initiates the radical polymerization that converts the liquid formulation to a solid, cured film. In our experiment, it was found that the ratio of the IRGACURE solutions played a critical role in the formation of the frit. When either IRGACURE 184 (αhydroxyketone) or IRGACURE 819 (bis acyl phosphine) as shown in Figure 2 was tested as the curing agent, the frit formation was failed. Since the manufacturer recommended the use of IRGACURE 184 in combination with IRGACURE

Site-selective immobilization of streptavidin on enzymatically biotinylated bacterial magnetic particles

AbstractBiotinylated 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)-streptavidin detection. 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.

Bioengineering of bacterial magnetic particles and its application to estrogen receptor-ligand binding assay

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.

One-step separation of CD20+cells from whole blood using bacterial magnetic particles displaying protein G

AbstractMagnetic separation of target cells from mixtures, such as peripheral blood and bone marrow, has considerable practical potential in research and medical applications. Among the current cell separation techniques, magnetic cell separation using immunomagnetic particles has been routinely applied and has proven rapidness and simplicity.Magnetospirillum magneticumAMB-1 synthesizes intracellular nano-sized bacterial magnetic particles (BacMPs) that are individually enveloped by a stable lipid bilayer membrane. BacMPs, which exhibit strong ferrimagnetism, can be collected easily with commercially available permanent magnets. In this study, a novel magnetic nanoparticle displaying protein G (protein G-BacMPs) was fabricated, and one-step cell separation for direct cell separation from whole blood was performed using the protein G-BacMPs. B lymphocytes (CD20+cells), which cover less than 0.3×10−2% of whole blood cells, were separated with 93% purity using protein G-BacMPs binding with anti-CD20 monoclonal antibodies. The results of this study demonstrate the utility of protein G-BacMPs and the magnetic cell separation approach based on protein G-BacMPs in numerous applications.

Bacterial magnetic particles (BMPs)‐PEI as a novel and efficient non‐viral gene delivery system

Non-viral methods of gene delivery, especially using polyethylenimine (PEI), have been widely used in gene therapy or DNA vaccination. However, the PEI system has its own drawbacks, which limits its applications. We have developed a novel non-viral delivery system based on PEI coated on the surface of bacterial magnetic nanoparticles (BMPs). The ability of BMPs-PEI complexes to bind DNA was determined by retardation of plasmid DNA in agarose gel electrophoresis. The transfection efficiency of BMPs-PEI/DNA complexes into eukaryotic cells was determined by flow cytometric analysis. The MTT assay was invited to investigate the cytotoxicity of BMPs-PEI/DNA complexes. The expression efficiency in vivo of BMPs-PEI bound to the plasmid pCMVbeta encoding beta-galactosidase was evaluated intramuscularly inoculated into mice. The immune responses of in vivo delivery of BMPs-PEI bound plasmid pcD-VP1 were determined by MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies. BMPs-PEI complexes could bind DNA and provide protection from DNase degradation. The transfection efficiency of BMPs-PEI/DNA complexes was higher than that in PEI/DNA complexes. Interestingly, in contrast to PEI, the BMPs-PEI complex was less cytotoxic to cells in vitro. We further demonstrated that the BMPs-PEI system can deliver an exogenous gene to animals and allow it to be expressed in vivo. Such expression resulted in higher levels of humoral and cellular immune responses against the target antigen compared to controls. We have developed a novel BMPs-PEI gene delivery system with a high transfection efficiency and low toxicity, which presents an attractive strategy for gene therapy and DNA vaccination.