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.     

Development of efficient expression system for protein display on bacterial magnetic particles

Bacterial magnetic particles (BMPs) are utilized for various biomedical applications because they are easily manipulated by magnets, and functional proteins are easily displayed on BMPs. To establish highly expressed protein display on BMPs, strong promoters were identified using Magnetospirillum magneticum AMB-1 genome and proteome databases. Initially, several proteins highly expressed in AMB-1 were identified, and the upstream DNA sequences of the open-reading frames were evaluated using a luciferase-reporter gene assay to compare promoter activities. Consequently, luminescence intensity was 400 times higher due to the novel promoter identified in this study than the magA promoter previously used. Subsequently, efficient protein display on BMPs was performed using the newly identified promoter sequences. This developed display system will facilitate the assembly of various functional proteins onto BMPs to create novel magnetic nanoparticles.     

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.     

Production of luciferase-magnetic particle complex by recombinant Magnetospirillum sp. AMB-1

Luciferase-bacterial magnetic particle (BMP) complexes were produced by recombinant Magnetospirillum sp. AMB-1. We constructed plasmids pKML and pNELM, respectively, by fusing luc to the 5' and 3' terminal of magA, encoding an integral iron translocating protein situated in the BMP membrane, of AMB-1. In addition, we produced bifunctional active-fusion proteins on BMPs by using a plasmid pAcML. In this plasmid, acetate kinase and luciferase genes were fused to the N-terminus and the C-terminus of MagA, respectively. Bacterial magnetic particles isolated from transconjugants for pKML, pNELM and pAcML exhibited luciferase activity. Bacterial magnetic particles isolated from transconjugants for pAcML also exhibited acetate kinase activity. Fed-batch culture of pKML transconjugant yielded 2.6 mg BMPs per liter of culture, and 95% conversion of iron into magnetite was obtained, at a nitrate concentration of 1.4 mM. Continuous feeding of iron as ferric quinate significantly enhanced growth and total magnetic production. Final cell concentration of 1.8 x 10(9) cells/mL and 6 mg per liter of culture was obtained. Magnetite production by fed-batch culture of AMB-1 was about 3 times that obtained by batch culture. There were no significant differences in BMPs yield between recombinant AMB-1 cultivated by fed-batch culture and wild type of AMB-1.     

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.     

Single nucleotide mismatch analysis using oligonucleotide probes synthesized on bacterial magnetic particle

An approach to analyze mismatches using short and specific oligonucleotide probes directly synthesized on bacterial magnetic particles (BMPs) by phosphoramidite methods was exploited. Approximately 126 molecules of 4-mer oligonucleotides/particle were synthesized on BMPs with high reaction efficiencies. Hybridization between FITC-labeled oligonucleotides and chemically synthesized oligonucleotides on BMPs was performed. Perfect matched and mismatched hybridizations were successfully discriminated by using the oligonucleotide probes on BMPs.     

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.     

Single nucleotide polymorphism genotyping of aldehyde dehydrogenase 2 gene using a single bacterial magnetic particle

A single nucleotide polymorphism (SNP) genotyping for aldehyde dehydrogenase 2 gene (ALDH2) has been developed by using a nano-sized magnetic particle, which was synthesized intracellularly by magnetic bacteria. Streptavidin-immobilized on bacterial magnetic particles (BMPs) were prepared using biotin labeled cross-linkers reacting with the amine group on BMPs. ALDH2 fragments from genomic DNA were amplified using a TRITC labeled primer and biotin labeled primer pair, and conjugated onto BMP surface by biotin-streptavidin interaction. PCR product-BMP complex was observed at a single particle level by fluorescence microscopy. These complexes were treated with restriction enzyme, specifically digesting the wild-type sequence of ALDH2 (normal allele of ALDH2). The homozygous (ALDH2*1/*1), heterozygous (ALDH2*1/*2), and mutant (ALDH2*2/*2) genotypes were discriminated by three fluorescence patterns of each particle. SNP genotyping of ALDH2 has been successfully achieved at a single particle level using BMP.     

Enhancement of magnetic particle production by nitrate and succinate fed-batch culture of Magnetospirillum sp. AMB-1

Summary Magnetospirillum sp. AMB-1 is a magnetic bacterium, which is capable of growing under air atmosphere. This bacterium was employed to make bacterial magnetic particles (BMPs). AMB-1 only makes BMPs during logarithmic growth phase under anaerobic conditions. Since it requires nitrate as a nitrogen source, control of nitrate concentration in the medium was necessary. The fed-batch culture of AMB-1 was carried out by adding nitric acid and succinate as nitrogen and carbon source respectively. One liter of AMB-1 culture produced 0.34 g of dry cells and 4.5 mg of BMPs. BMP production by AMB-1 cultivated in the fed-batch culture was found to be seven times higher than that cultivated in the batch culture.     

Use of magnetic carboxyl beads to purify a cationic peptide in a batch system

Antimicrobial peptides (AMPs) are cationic molecules that are good leads for new antiinfective drugs. To obtain sufficient amounts, recombinant AMPs are generally produced as fusion proteins in Escherichia coli. Fusion partners facilitate purification of recombinant proteins. Fusion proteins are then cleaved by specific proteases, and cationic peptides are purified by size exclusion chromatography or ion exchange chromatography, neither of which is easily applicable to small volumes of diluted peptide samples. We developed a small-scale system that is easily adaptable for high-throughput screening and uses carboxyl magnetic beads to purify a cationic peptide from its fusion partner.     

Novel detection system for biomolecules using nano-sized bacterial magnetic particles and magnetic force microscopy

A system for streptavidin detection using biotin conjugated to nano-sized bacterial magnetic particles (BMPs) has been developed. BMPs, isolated from magnetic bacteria, were used as magnetic markers for magnetic force microscopy (MFM) imaging. The magnetic signal was obtained from a single particle using MFM without application of an external magnetic field. The number of biotin conjugated BMPs (biotin-BMPs) bound to streptavidin immobilized on the glass slides increased with streptavidin concentrations up to 100 pg/ml. The minimum streptavidin detection limit using this technique is 1 pg/ml, which is 100 times more sensitive than a conventional fluorescent detection system. This is the first report using single domain nano-sized magnetic particles as magnetic markers for biosensing. This assay system can be used for immunoassay and DNA detection with high sensitivities.     

Detection of HbA(1c) by boronate affinity immunoassay using bacterial magnetic particles.

We have developed a boronate affinity immunoassay system using m-aminophenylboronic acid (mAPB) coupling to bacterial magnetic particles (BMPs). Homobifunctional crosslinker, Bis-(succcimidyl)suberate (BS3), was employed for preparation of mAPB-BMPs conjugates (mAPB-BMPs). Quantities of HbA_1c on mAPB-BMPs were evaluated based on luminescence from alkaline phosphatase-conjugated anti-Hb antibody (ALP–antibody) binding to HbA_1c on the BMP surface. The binding of HbA_1c to mAPB-BMPs occurred gradually and was almost completed within 10 mm. The coupling reaction is enhanced due to static electric interaction between the positive charges on HbA_1c and negative charges on BMPs. The amount of HbA_1c binding to mAPB-BMPs increased with increasing sodium chloride concentrations in the range of 0–100 mM. However, the amount of Hb binding to mAPB-BMPs also increased in high concentration of sodium chloride. The Hb binding to mAPB-BMPs was detached from mAPB-BMPs when Hb–mAPB-BMPs were washed with low salt buffer. This indicates that Hb is nonspecifically adsorbed onto the surface of mAPB-BMPs in high concentration of sodium chloride. These results suggest that selective separation of HbA_1c using mAPB-BMPs can be achieved with these conditions. A dose–response curve was obtained between luminescence intensity and HbA_1c concentration using a fully automated boronate affinity immunoassay. A linear relationship between luminescence intensity and HbA_1c concentration was obtained in the range of 10–10⁴ ng/ml.     

基于谷胱甘肽-磁性微粒的GST融合蛋白纯化体系的建立

本研究将还原型谷胱甘肽(GSH)共价结合在异硫氰酸根末端磁性微粒表面,制备了具有超顺磁性的谷胱甘肽-磁性微粒亲和介质,以表面修饰有谷胱甘肽的磁性微粒为载体,建立了谷胱甘肽巯基转移酶(GST)融合蛋白的纯化体系。对100μL细胞裂解液纯化体系所需磁性微粒用量、谷胱甘肽-磁性微粒与细胞裂解液的孵育时间、清洗条件等进行了优化。以聚丙烯酰胺凝胶电泳对融合蛋白的纯度进行了检测,Bradford方法对融合蛋白进行了定量测定,对纯化得到的目的蛋白进行了Western blotting分析。结果表明,每毫克异硫氰酸根末端磁性微粒对GSH的固定化容量为150μg,10 mg谷胱甘肽-磁性微粒可满足100μL细胞裂解体系中目的蛋白的纯化,最佳孵育时间为40 min,对GST融合蛋白的平均纯化量为516μg。本方法快速、简便,基于磁性微粒的分离还可实现自动化,对GST融合蛋白的纯化具有很好的应用前景。     

Detection of HbA1c by boronate affinity immunoassay using bacterial magnetic particles

We have developed a boronate affinity immunoassay system using m-aminophenylboronic acid (mAPB) coupling to bacterial magnetic particles (BMPs). Homobifunctional crosslinker, Bis-(succcimidyl)suberate (BS3), was employed for preparation of mAPB-BMPs conjugates (mAPB-BMPs). Quantities of HbA_1c on mAPB-BMPs were evaluated based on luminescence from alkaline phosphatase-conjugated anti-Hb antibody (ALP–antibody) binding to HbA_1c on the BMP surface. The binding of HbA_1c to mAPB-BMPs occurred gradually and was almost completed within 10 mm. The coupling reaction is enhanced due to static electric interaction between the positive charges on HbA_1c and negative charges on BMPs. The amount of HbA_1c binding to mAPB-BMPs increased with increasing sodium chloride concentrations in the range of 0–100 mM. However, the amount of Hb binding to mAPB-BMPs also increased in high concentration of sodium chloride. The Hb binding to mAPB-BMPs was detached from mAPB-BMPs when Hb–mAPB-BMPs were washed with low salt buffer. This indicates that Hb is nonspecifically adsorbed onto the surface of mAPB-BMPs in high concentration of sodium chloride. These results suggest that selective separation of HbA_1c using mAPB-BMPs can be achieved with these conditions. A dose–response curve was obtained between luminescence intensity and HbA_1c concentration using a fully automated boronate affinity immunoassay. A linear relationship between luminescence intensity and HbA_1c concentration was obtained in the range of 10–10⁴ ng/ml.     

Capture and release of DNA using aminosilane-modified bacterial magnetic particles for automated detection system of single nucleotide polymorphisms

Bacterial magnetic particles (BMPs) were modified with 3-[2-(2-aminoethylamino)-ethylamino]-propyltrimethoxysilane (AEEA) to produce a dense amine surface. Modification of BMPs in a toluene solution resulted in an increased amine yield, and approximately 11.3 x 10(4) surface amines were detected on a single particle. The modified BMPs were capable of efficient electrostatic capture of DNA. The maximum amount of DNA captured on 10 microg of aminosilane-modified BMPs was 600 ng. A 10 mM phosphate buffer effectively released the captured DNA. This efficiency was dramatically enhanced by incubation at 80 degrees C and DNA recovery from aminosilane-modified BMPs approached 95%. DNA extraction from whole blood using these modified BMPs, followed by PCR, was successfully performed. Furthermore, automated single nucleotide polymorphism (SNP) detection of the aldehyde dehydrogenase 2 (ALDH2) was demonstrated.     

Trypanosoma cruzi: fusogenic ability of membranes from cultured epimastigotes in interaction with human syncytiotrophoblast

Trypanosoma cruzi epimastigotes cultured in vitro were disrupted by successive freezing and thawing and subsequent sonication. The total homogenate was fractionated by differential centrifugation to obtain an enriched plasma membrane fraction. The proteins of subcellular parasite fractions were labeled with 131I and their binding to membrane fractions from human placenta syncytiotrophoblast was studied. Syncytiotrophoblast fractions enriched in plasma showed higher specific activity for binding an enriched T. cruzi plasma membrane fraction compared with other fractions of syncytiotrophoblast. The properties of this interaction were studied with digestive enzymes (trypsin and phospholipase A2). The results showed that both proteins and lipids could be involved in this interaction. The Ca2+ requirements for the membrane-membrane interaction are different for the two membranes studied. Also the enriched plasma membrane T. cruzi fraction had a higher capacity to induce fusion processes than the other subcellular fractions. The above results indicate that a preferential syncytiotrophoblast-T. cruzi interaction may occur between the two cell surfaces as compared to intracellular membranes and that the parasite surface is able to induce an instability process leading to membrane fusion. These results may have implications in regard to the mechanism of entry of the parasite into cells.     

Isolation of nuclear encoded plastid ribosomal protein cDNAs

Summary A pea leaf cDNA library was constructed in the expression vector gt11 and screened with antisera raised against proteins extracted from 30S and 50S ribosomal subunits and 70S ribosomes prepared from isolated pea chloroplasts. Six recombinant phage were identified that encoded fusion proteins containing plastid ribosomal protein antigenic determinants. Phage-induced cell lysate proteins, containing the fusion proteins, were bound to nitrocellulose membranes and used as affinity matrices to prepare monospecific antibodies. These antibodies were then used to identify by Western blotting which plastid ribosomal protein shared antigenic determinants with the fusion proteins. cDNA inserts from the antigen-producing phage were used to hybrid-select complementary mRNAs. The cell-free translation products of these mRNAs were added to a pea chloroplast in vitro transport system and imported proteins analyzed by two-dimensional gel electrophoresis. The imported proteins comigrated with the plastid ribosomal proteins that were identified as being antigenically related to the fusion proteins produced by the corresponding recombinant phage. The imported proteins were 3,500–5,500 daltons smaller than their precursors.     

Mutagenesis and nuclear magnetic resonance analyses of the fusion peptide of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus F protein

The entry of enveloped viruses into cells is normally mediated by fusion between viral and cellular membranes, in which the fusion peptide plays a crucial role. The fusion peptides of group II nucleopolyhedrovirus (NPV) F proteins are quite conserved, with a hydrophobic region located at the N terminal of the F(1) fragment. For this report, we used mutagenesis and nuclear magnetic resonance (NMR) to study the structure and function of the fusion peptide of the Helicoverpa armigera single-nucleocapsid NPV (HearNPV) F protein (HaF). Five mutations in the fusion peptide of HaF, N(1)G, N(1)L, I(2)N, G(3)L, and D(11)L, were generated separately, and the mutated f genes were transformed into the f-null HearNPV bacmid. The mutations N(1)L, I(2)N, and D(11)L were found to completely abolish the ability of the recombinant bacmids to produce infectious budded virus, while the mutations N(1)G and G(3)L did not. The low-pH-induced envelope fusion assay demonstrated that the N(1)G substitution increased the fusogenicity of HaF, while the G(3)L substitution reduced its fusogenicity. NMR spectroscopy was used to determine the structure of a synthetic fusion peptide of HaF in the presence of sodium dodecyl sulfate micelles at pH 5.0. The fusion peptide appeared to be an amphiphilic structure composed of a flexible coil in the N terminus from N(1) to N(5), a 3(10)-helix from F(6) to G(8), a turn at S(9), and a regular alpha-helix from V(10) to D(19). The data provide the first NMR structure of a baculovirus fusion peptide and allow us to further understand the relationship of structure and function of the fusion peptide.     

An optimized ultrasonication protocol for bacterial cell disruption and recovery of β-galactosidase fusion proteins

Summary In this work, we have developed and optimized an ultrasonication protocol for Escherichia coli recombinant cells, adapted to laboratory-scale release of -galactosidase fusion proteins. After a single sonication treatment of 15 minutes, about 30% of recombinant protein present in the sample remains still associated to cellular debris, and it can not be removed by increasing the sonication time. After a clarification step a second sonication treatment of the resuspended cell debris again releases only a 70% of the remaining product. Therefore, the application of two short, consecutive sonication treatments permits a global recovery yield of about 90%. The use of a new disruption buffer to stabilize -galactosidase allows the fusion proteins to maintain the active form throughout the process.