氨基末端磁性载体固定化中性蛋白酶的研究

以氨基末端磁微粒为载体,用戊二醛作交联剂,通过共价交联结合法固定化AS1.398中性蛋白酶.可以制备出活力达45 000 U/g磁性固定化酶.探讨了该载体对中性蛋白酶的最适固定化条件,并对磁性固定化酶的热稳定性,储存稳定性、操作稳定性等进行了研究,确定了此载体对酶的固载能力大于200 mg/g（载体）,及固定化磁性酶最适pH为7.5, 最适温度为60℃等催化特性.     

Preparation of Fine Magnetic Particles and Application for Enzyme Immobilization

Fine magnetic particles (ferrofluid) were prepared from a co-precipitation method by oxidation of Fe²⁺ with nitrite. The particles were activated with (3-aminopropyl)triethoxysilane in toluene and the activated particles were combined with some enzymes by using glutaraldehyde. Enzyme-immobilized magnetic particles were between 4-70 nm and the size could be changed corresponding to the ratio of the amount of Fe²⁺ to that of nitrite. In the immobilization of β-glucosidase, activity yield was 83% and 168 mg protein was immobilized per g magnetite. Other enzymes or proteins could be immobilized at the level between about 70 and 200mg/g support. Immobilized β-glucosidase was stable at 4°C. Magnetic particles immobilized with β-glucosidase responded quickly to the magnetic field and “ON-OFF” control of the enzyme reaction was possible.     

Preparation of Fine Magnetic Particles and Application for Enzyme Immobilization

Fine magnetic particles (ferrofluid) were prepared from a co-precipitation method by oxidation of Fe²⁺ with nitrite. The particles were activated with (3-aminopropyl)triethoxysilane in toluene and the activated particles were combined with some enzymes by using glutaraldehyde. Enzyme-immobilized magnetic particles were between 4-70 nm and the size could be changed corresponding to the ratio of the amount of Fe²⁺ to that of nitrite. In the immobilization of β-glucosidase, activity yield was 83% and 168 mg protein was immobilized per g magnetite. Other enzymes or proteins could be immobilized at the level between about 70 and 200mg/g support. Immobilized β-glucosidase was stable at 4°C. Magnetic particles immobilized with β-glucosidase responded quickly to the magnetic field and “ON-OFF” control of the enzyme reaction was possible.     

A Comparison of In Vitro- and In Vivo Phosphorylated Neurofilament Polypeptides

Abstract: Neurofilament polypeptides phosphorylated in vitro by incubation of neurofilament-enriched preparations from rat CNS with [γ-³²P]ATP were compared with the corresponding polypeptides labeled in vivo by injection of ³²P_i into the lateral ventricles of rats. Autoradiography of sodium dodecyl sulfate (SDS)-polyacrylamide gels revealed that the major phosphorylated species in both preparations were the three neurofilament subunits, which have molecular weights of 200K, 145K, and 68K. However, the relative levels of ³²P detected in the three in vitro -labeled subunits differed from the relative in vivo levels. The two larger neurofilament polypeptides displayed similar ³²P isoprotein distribution patterns on two-dimensional gels, whereas additional isoproteins were seen in the in vitro -labeled 68K species. Limited proteolysis in SDS-polyacrylamide gels revealed the presence of common phosphopeptides in the corresponding pairs of in vitro- and in vivo-labeled subunits, but the in vivo -labeled 145K and in vitro -labeled 200K polypeptides contained additional digestion products. Two-dimensional peptide mapping of the 68K polypeptide digested with a mixture of trypsin and chymotrypsin indicated that this component was phosphorylated at a single, identical site, both in vivo and in vitro. These results indicate that the protein kinase that copurifies with neurofilament preparations may be involved in their in vivo phosphorylation.     

磁细菌与细菌磁颗粒的特点及应用研究

概述了磁细菌的特点及由磁细菌所产生的细菌磁颗粒的晶体成分、形态特征、磁颗粒膜的特点以及细菌磁颗粒在信息贮存、磁性细胞制备、基因研究、生物活性物质载体、免疫检测以及在污水处理、矿物分选等方面的应用研究。     

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-I.With this method,a very low minimum antigen concentration (1 x 106 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 (106) higher than an ELISA-based method of detection PNRSV and GFLV.     

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 （1×10^6 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.     

In Vivo Biotinylation of Bacterial Magnetic Particles by a Truncated Form of Escherichia coli Biotin Ligase and Biotin Acceptor Peptide

Escherichia coli biotin ligase can attach biotin molecules to a lysine residue of biotin acceptor peptide (BAP), and biotinylation of particular BAP-fused proteins in cells was carried out by coexpression of E. coli biotin ligase (in vivo biotinylation). This in vivo biotinylation technology has been applied for protein purification, analysis of protein localization, and protein-protein interaction in eukaryotic cells, while such studies have not been reported in bacterial cells. In this study, in vivo biotinylation of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1 was attempted by heterologous expression of E. coli biotin ligase. To biotinylate BacMPs in vivo, BAP was fused to a BacMP surface protein, Mms13, and E. coli biotin ligase was simultaneously expressed in the truncated form lacking the DNA-binding domain. This truncation-based approach permitted the growth of AMB-1 transformants when biotin ligase was heterologously expressed. In vivo biotinylation of BAP on BacMPs was confirmed using an alkaline phosphatase-conjugated antibiotin antibody. The biotinylated BAP-displaying BacMPs were then exposed to streptavidin by simple mixing. The streptavidin-binding capacity of BacMPs biotinylated in vivo was 35-fold greater than that of BacMPs biotinylated in vitro, where BAP-displaying BacMPs purified from bacterial cells were biotinylated by being mixed with E. coli biotin ligase. This study describes not only a simple method to produce biotinylated nanomagnetic particles but also a possible expansion of in vivo biotinylation technology for bacterial investigation.Biotin/streptavidin binding is the strongest noncovalent interaction known in nature (K_d [dissociation constant], ∼10⁻¹⁵ M) (10), and this tight binding is one of the most general tools for biological research and has been widely used for biomolecular detection (11, 12), immobilization (14, 19), and recovery (15). Therefore, it is of great significance to biotinylate biomolecules, in particular, proteins without functional inhibition. For this purpose, the method for site-selective biotinylation of proteins had been developed using biotin ligase. Biotin ligase catalyzes the posttranslational biotinylation of biotin enzymes, such as acetyl coenzyme A (acetyl-CoA) carboxylase, and introduces biotin into a specific lysine residue of a biotin carboxyl carrier protein (BCCP), a subunit of biotin enzymes (13). In early studies, BCCP (∼100 amino acid residues) had been fused with the proteins of interest for biotinylation by biotin ligase (7); however, there was a concern that fused BCCP might disrupt the function of target proteins. Recently, biotin acceptor peptides (BAPs) had replaced BCCP due to the advantage of small size. BAPs, with 15 to 23 amino acid residues, were screened from a peptide library as peptide tags biotinylated by Escherichia coli biotin ligase (4, 25). BAP-fused proteins can be biotinylated outside the cells by adding biotin and purified E. coli biotin ligase with Mg²⁺ and ATP (in vitro biotinylation). Furthermore, it is also possible to biotinylate BAP-fused proteins inside the cells with coexpression of E. coli biotin ligase (in vivo biotinylation) because BAP is specifically recognized only by E. coli biotin ligase. This in vivo biotinylation technology has been applied in eukaryotic cells to purify the proteins by using streptavidin-immobilized resin (8, 24, 28), because biotin/streptavidin interaction permits stringent washing to eliminate the nonspecific binding. Specific biotinylation can be applied also for protein localization analysis. Using fluorophore- or gold nanoparticle-labeled streptavidin, biotinylated proteins were clearly observed in a previous study (27). Recently, a novel technique to detect protein-protein interaction by fusing BAP and biotin ligase was developed by Ting''s group. BAP and biotin ligase were fused to different two proteins, and then the interaction of these proteins was successfully evaluated via biotinylation of BAP (9). In vivo biotinylation technology using heterologously expressed E. coli biotin ligase should be equally useful for prokaryotes; however, such studies have not been reported for bacterial cells.Magnetospirillum magneticum AMB-1, a magnetotactic bacterium, synthesizes intracellular nanosized bacterial magnetic particles (BacMPs) of 50 to 100 nm; these are surrounded by a lipid bilayer membrane, possess a single magnetic domain of magnetite, and exhibit strong ferrimagnetism (18). Furthermore, functional proteins have been displayed on BacMP surfaces through gene fusion techniques (21, 30, 31). BacMP membrane proteins, including Mms13, were used as anchor proteins; this approach permits functional proteins to be localized efficiently and oriented appropriately on BacMPs (31). We recently reported a novel method for the simple production of biotin-labeled magnetic particles through protein display techniques, where introduction of the biotin moiety onto BacMPs was carried out by the endogenous biotin ligase (17). For the biotinylation of BacMPs, we screened the gene encoding BCCP in the AMB-1 genome and displayed it on the surface of BacMPs using an anchor protein, Mms13. BCCP-displaying BacMPs were biotinylated by endogenous AMB-1 biotin ligase in the cells with high efficiency. This in vivo modification approach could be applied for construction of BacMP-quantum dot nanocomposites toward multicolor labeling of cancer cells, where BCCP and antibody carrier protein (protein G) were simultaneously displayed in tandem (16). However, the size of BCCP, with 149 amino acid residues and a mass of 15.6 kDa, makes it rather large for use as a labeling tag. Although it would be preferable to use a smaller peptide, BAP, for the tag to minimize effects on the flanking proteins for future applications, BAP was not recognized and biotinylated by endogenous AMB-1 biotin ligase (17).In this study, in vivo biotinylation of BacMPs was attempted by heterologous expression of E. coli biotin ligase and Mms13-BAP fusion protein in AMB-1 cells. First, the method for effective expression of E. coli biotin ligase in bacterial cells was optimized. Then site-selective biotinylation of BAP on BacMPs was confirmed. Finally, the obvious advantage of in vivo biotinylation of BAP-displaying BacMPs compared with the in vitro biotinylation method was demonstrated.     

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.Transmembrane proteins play critical roles in cellular metabolism, participating in processes such as ion transport, nutrient uptake, signal transduction, and intercellular communication. As evidence of the essential functions of these proteins, more than half of all drug targets have been shown to be transmembrane proteins, and the analysis of the interactions of transmembrane proteins and their ligands is one of the most promising avenues for the discovery of new drug candidates. As a means of producing sufficient amounts of transmembrane proteins for binding analyses, heterologous protein expression systems have been developed using Escherichia coli (10), yeast (16), insect, and mammalian (4) cells as hosts. Transmembrane proteins generally are expressed at low levels and are extremely hydrophobic, rendering the analysis of interactions with ligands very difficult. In all cases, the analysis of membrane proteins requires a lipid or similar synthetic environment to maintain the native structure and function of the proteins. The purification of transmembrane proteins from cells frequently is time-consuming and typically results in the loss of the proteins’ native conformation.Magnetospirillum magneticum AMB-1 synthesizes intracellular nanosized bacterial magnetic particles (BacMPs; 50 to 100 nm); these are surrounded by a lipid bilayer membrane and exhibit strong ferrimagnetism. Functional soluble proteins have been expressed on BacMP surfaces through gene fusion techniques (11, 21, 24, 27) using BacMP membrane proteins (MagA, Mms16, and Mms13) as anchor proteins; this approach permits heterologous proteins to be localized efficiently and oriented appropriately on BacMPs. In a previous report, we demonstrated the successful display of the D1 dopamine receptor, a G protein-coupled receptor possessing seven transmembrane domains, on BacMPs. Mms16-D1, an dopamine receptor fusion protein, was expressed under the mms16 promoter, and a ligand-binding assay was performed (28). The assembly of transmembrane proteins on magnetic particles provides significant advantages for binding assays, including the easing of the purification of target proteins from bacterial cells without the loss of native conformation and the availability of a fully automated bioassay using robotic magnetic separation. Despite these advantages, there are not enough studies for the overexpression of transmembrane proteins other than the D1 dopamine receptor in M. magneticum AMB-1 because of its difficulty. In other host cells, a system for controlling gene expression has been employed to overcome its difficulty, and some successful efforts had achieved this for crystal structure analysis (5, 15, 18). The lack of these systems for M. magneticum has hampered the extension of this application to other transmembrane proteins.In this study, the tetracycline-inducible expression system was adapted for displaying transmembrane proteins on BacMPs in M. magneticum AMB-1. Expression vectors carrying the tetracycline repressor gene (tetR) and the target gene under the control of a strong promoter and the tetracycline operator (tetO) sequence were constructed, and the function of the system was evaluated using reporter genes. Finally, this system was applied to the overexpression of the transmembrane protein, tetraspanin CD81. This is the first report of an inducible expression system in M. magneticum, and it the demonstrates efficient display of a transmembrane protein at the surface of BacMPs.     

In Vivo Self-Assembly of Stable Green Fluorescent Protein Fusion Particles and Their Uses in Enzyme Immobilization

Bacterial inclusion bodies are aggregations of mostly inactive and misfolded proteins. However, previously the in vivo self-assembly of green fluorescent protein (GFP) fusions into fluorescent particles which displayed specific binding sites suitable for applications in bioseparation and diagnostics was demonstrated. Here, the suitability of GFP particles for enzyme immobilization was assessed. The enzymes tested were a thermostable α-amylase from Bacillus licheniformis, N-acetyl-d-neuraminic acid aldolase (NanA) from Escherichia coli, and organophosphohydrolase (OpdA) from Agrobacterium radiobacter. Respective GFP particles were isolated and could be stably maintained outside the cell. These enzyme-bearing GFP particles exhibited considerable stability across a range of temperature, pH, and storage conditions and could be recycled. The α-amylase-bearing particles retained activity after treatments at 4 to 85°C and at pHs 4 to 10, were stable for 3 months at 4°C, and could be recycled up to three times. OpdA-bearing particles retained degradation activity after treatments at 4 to 45°C and at pHs 5 to 10 and were able to be recycled up to four times. In contrast, the performance of NanA-bearing particles rapidly declined (>50% loss) after each recycling step and 3 months storage at 4°C. However, they were still able to convert N-acetylmannosamine and pyruvate to N-acetylneuraminic acid after treatment at 4 to 85°C and at pHs 4 to 11. Fluorescent GFP fusion particles represent a novel method for the immobilization and display of enzymes. Potential applications include diagnostic assays, biomass conversion, pharmaceutical production, and bioremediation.     

Efficient and Stable Display of Functional Proteins on Bacterial Magnetic Particles Using Mms13 as a Novel Anchor Molecule

Magnetic particles are increasingly used for various biomedical applications because they are easy to handle and separate from biological samples. In this work, a novel anchor molecule was used for targeted protein display onto magnetic nanoparticles. The magnetic bacterium Magnetospirillum magneticum AMB-1 synthesizes intracellular bacterial magnetic particles (BMPs) covered with a lipid bilayer membrane. In our recent research, an integral BMP membrane protein, Mms13, was isolated and used as an anchor molecule to display functional proteins onto BMPs. The anchoring properties of Mms13 were confirmed by luciferase fusion studies. The C terminus of Mms13 was shown to be expressed on the surface of BMPs, and Mms13 was bound to magnetite directly and tightly permitting stable localization of a large protein, luciferase (61 kDa), on BMPs. Consequently, luminescence intensity obtained from BMPs using Mms13 as an anchor molecule was >400 or 1,000 times higher than Mms16 or MagA, which previously were used as anchor molecules. Furthermore, the immunoglobulin G-binding domain of protein A (ZZ) was displayed uniformly on BMPs using Mms13, and antigen was detected by transmission electron microscopy using antibody-labeled gold nanoparticles on a single BMP displaying the ZZ-antibody complex. The results of this study demonstrated the utility of Mms13 as a molecular anchor, which will facilitate the assembly of other functional proteins onto BMPs in the near feature.     

葡聚糖磁性毫微粒固定化L－天冬酰胺酶的研究

葡聚糖磁性毫微粒固定化Ｌ－天冬酰胺酶的研究徐慧显,李民勤,潘再群,马建标,何炳林（南开大学高分子化学研究所,天津３０００７１）大肠杆菌天冬酰胺酶对急性淋巴白血病有明显疗效［１］,注射入体内以后,可迅速清除血清中的天冬酰胺──敏感性肿瘤细胞的必需营养成...     

氨基化二氧化硅颗粒固定木瓜蛋白酶研究

采用正硅酸乙酯与N-(β-氨乙基)氨丙基三乙氧基硅烷在油包水形成的微胶囊中同步水解的方法,一步法制备了氨基化的二氧化硅颗粒,得到的颗粒粒径在0.3～0.5μm之间,平均大小为0.37μm, 氨基含量和颗粒大小可控,氨基含量高达56mmol/g。此颗粒经戊二醛处理后,采用共价法固定木瓜蛋白酶,固定化最适pH6.5,最佳给酶量为15mg/g载体,固定化酶的最适反应温度为70℃,最适反应pH为6.5,固定化酶热稳定性,pH耐受性,贮存稳定性都明显高于游离酶,表明此颗粒可作为一种优良的酶固定化载体。     

Bacterial Colonization of Particles: Growth and Interactions

Marine particles in the ocean are exposed to diverse bacterial communities, and colonization and growth of attached bacteria are important processes in the degradation and transformation of the particles. In an earlier study, we showed that the initial colonization of model particles by individual bacterial strains isolated from marine aggregates was a function of attachment and detachment. In the present study, we have investigated how this colonization process was further affected by growth and interspecific interactions among the bacteria. Long-term incubation experiments showed that growth dominated over attachment and detachment after a few hours in controlling the bacterial population density on agar particles. In the absence of grazing mortality, this growth led to an equilibrium population density consistent with the theoretical limit due to oxygen diffusion. Interspecific interaction experiments showed that the presence of some bacterial strains (“residents”) on the agar particles either increased or decreased the colonization rate of other strains (“newcomers”). Comparison between an antibiotic-producing strain and its antibiotic-free mutant showed no inhibitory effect on the newcomers due to antibiotic production. On the contrary, hydrolytic activity of the antibiotic-producing strain appeared to benefit the newcomers and enhance their colonization rate. These results show that growth- and species-specific interactions have to be taken into account to adequately describe bacterial colonization of marine particles. Changes in colonization pattern due to such small-scale processes may have profound effects on the transformation and fluxes of particulate matter in the ocean.     

氨基磁性微球固定化木瓜蛋白酶及其应用

目的:采用实验室自制氨基磁性微球固定化木瓜蛋白酶,并将其应用于啤酒中蛋白质的水解.方法:考察不同给酶量、戊二醛浓度、固定时间等因素对固定化木瓜蛋白酶的影响.将固定化酶用于啤酒的处理,测定其游离氨基酸变化.结果:实验表明给酶量1.2mg/ml,戊二醛浓度5%,固定化时间4h为固定化最佳条件.酶活力测定表明,在最优化条件下制备的固定化酶较溶液酶具有更低的Km值(0.489%:3.412%),更好的酸碱(6.8:7.2)和温度耐受性(77℃:67℃),操作和储藏稳定性也得到了很大提高.固定化酶水解啤酒中蛋白质后,啤酒混浊度降低了0.013个单位,酪氰酸和苯丙氨酸等游离氨基酸有不同程度的增加,啤酒色泽和口味得到了改善.结论:磁性固定化木瓜蛋白酶较溶液酶有更佳的稳定性和更好的可操作性,具有广泛的应用前景.     

磁性复合微球固定化酶制备过程及其研究进展

磁性复合微球作为一种优良的载体,广泛应用于生物医学和技术上,如蛋白纯化、药物绑定、酶固定化等.磁性复合微球制备过程包括纳米磁性粒子合成、磁性复合微球制备,将酶与经表面戎基、氛基、环氧基等功能基团修饰或直接与磁性微球共价结合,或者与表面经金属离子鳌合的磁性微球吸附从而实现酶固定化.本文介绍了磁性复合微球的制备过程及其在固定化酶方面的研究进展.     

Assembly of G Protein-Coupled Receptors onto Nanosized Bacterial Magnetic Particles Using Mms16 as an Anchor Molecule

G protein-coupled receptors (GPCRs) play a central role in a wide range of biological processes and are prime targets for drug discovery. GPCRs have large hydrophobic domains, and therefore purification of GPCRs from cells is frequently time-consuming and typically results in loss of native conformation. In this work, GPCRs have been successfully assembled into the lipid membrane of nanosized bacterial magnetic particles (BMPs) produced by the magnetic bacterium Magnetospirillum magneticum AMB-1. A BMP-specific protein, Mms16, was used as an anchor molecule, and localization of heterologous Mms16 on BMPs was confirmed by luciferase fusion studies. Stable luminescence was obtained from BMPs bearing Mms16 fused with luciferase at the C-terminal region. D1 dopamine receptor (D1R), a GPCR, was also efficiently assembled onto BMPs by using Mms16 as an anchor molecule. D1R-BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants. After washing, the complexes were ready to use for analysis. This system conveniently refines the native conformation of GPCRs without the need for detergent solubilization, purification, and reconstitution after cell disruption.     

链霉亲和素纯化和鉴定方法的研究

对链霉亲和素进行纯化、鉴定,采用冷钝化的方法去除培养液中大部分杂蛋白,用亲和层析法从链霉菌L-183的培养液中纯化链霉亲和素（SA）,经试验,SA回收率为75％～85％。鉴定表明,自制SA的分子量为74．5kD,每分子SA可结合3．2个生物素分子,活性为11．2U／mg,pI为7．4。自制SA各项生物学性质与文献报道相符。     

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 × 10¹⁰ PFU of the library phage (complexity, 2.7 × 10⁹) 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.