Immunostick colorimetric assay for highly sensitive detection of food allergens by bio-nanocapsule-scaffolding technology.

The detection sensitivity of immunostick colorimetric assay has been increased by using a bio-nanocapsule as a scaffold for oriented immobilization of immunoglobulin Gs. This immunostick produced ∼82-folds stronger coloration in the detection of food allergens and reduced detection time by a factor of 5.

Mass-Fabrication Scheme of Highly Sensitive Wireless Electrodeless MEMS QCM Biosensor with Antennas on Inner Walls of Microchannel.

Quartz-crystal-microbalance (QCM) biosensor is a typical label-free biosensor, and its sensitivity can be greatly improved by removing electrodes and wires that would be otherwise attached to the surfaces of the quartz resonator. The wireless-electrodeless QCM biosensor was then developed using a microelectro-mechanical systems (MEMS) process, although challenges remain in the sensitivity, the coupling efficiency, and the miniaturization (or mass production). In this study, we establish a MEMS process to obtain a large number of identical ultrasensitive and highly efficient sensor chips with dimensions of 6 mm square. The fundamental shear resonance frequency of the thinned AT-cut quartz resonator packaged in the microchannel exceeds 160 MHz, which is excited by antennas deposited on inner walls of the microchannel, significantly improving the electro-mechanical coupling efficiency in the wireless operation. The high sensitivity of the developed MEMS QCM biosensors is confirmed by the immunoglobulin G (IgG) detection using protein A and ZZ-tag displaying a bionanocapsule (ZZ-BNC), where we find that the ZZ-BNC can provide more effective binding sites and higher affinity to the target molecules, indicating a further enhancement in the sensitivity of the MEMS QCM biosensor. We then perform the label-free C-reactive protein (CRP) detection using the ZZ-BNC-functionalized MEMS QCM biosensor, which achieves a detection limit of 1 ng mL-1 or less even with direct detection.

Nano-visualization of the in vitro antiviral activity of black tea based on production area using a liposome-based virus membrane model.

Black tea extracts (BTEs) from four different production areas showed a higher aggregation strength for phosphatidylcholine-based liposomes containing cholesterol used as a viral membrane model. Furthermore, the anti-influenza A virus (IAV) activity of each BTE in vitro demonstrated that although Sri Lanka, Kenya, and Assam had higher anti-IAV activities, Darjeeling had a lower anti-IAV activity, showing a correlation between each BTE and the liposome aggregation strength. Moreover, the antiviral activity strength of BTEs was consistent with the antioxidant activity strength of BTEs, suggesting that the component(s) in black tea that exhibits antioxidant activity would also be the component(s) that accounts for its antiviral activity. Thus, our results propose that BTEs exert their antiviral effects by binding not only hemagglutinin and neuraminidase but also viral membranes directly, especially "cholesterol-rich lipid rafts" and affect the membrane structure, causing the virus to aggregate, thereby inhibiting infection of the host cells.

Mechanical detection of interactions between proteins related to intermediate filament and transcriptional regulation in living cells.

Intermediate filaments (IF) bind to various proteins and regulate cell function in the cytoplasm. Recently, IFs were found to regulate gene expression by acting as capture scaffolds for transcription-related proteins and preventing their translocation into the nucleus. To reveal such transcriptional regulatory mechanisms controlled by IFs, a method to analyze the interaction between IFs and transcription-related proteins is necessary. Although there are many methods to observe interactions in living cells, it is still challenging to measure protein-protein interactions in living cells in their unmodified and native state. In this study, we utilized a nanoneedle that can access the cytosol by insertion into the cell. Modification of antibody recognizing transcription-related proteins allows the needle to detect mechanical force required to unbind the interaction between antibody and target proteins interacting with IFs during retraction of the needle from the cell. We focused on IF vimentin, a marker of epithelial-mesenchymal transition, to mechanically detect transcription-related proteins trapped by vimentin filaments. Prohibitin 2 (PHB2), a transcription-related factor, was selected as the candidate vimentin-binding protein. We conducted mechanical detection of PHB2 using atomic force microscopy and anti-PHB2 antibody-modified nanoneedles in vimentin-expressing mouse breast cancer and vimentin-knockout (VKO) cells. Significantly larger unbinding forces were detected in the vimentin-expressing cells than in the VKO cells. The results demonstrate that this method is useful for in-cell mechanical detection of IF-binding proteins.

Bio-nanocapsules for oriented immobilization of DNA aptamers on aptasensors.

The oriented immobilization of sensing molecules (e.g., IgGs, receptors, lectins, and DNA aptamers) on sensor chips is particularly important for maximizing the potential of the sensing molecules, thereby enhancing the sensitivity and target-binding capacity of biosensors. We previously developed ∼30 nm bio-nanocapsules (ZZ-BNCs) consisting of the hepatitis B virus envelope L protein fused with the tandem form of protein A-derived IgG Fc-binding Z domain (ZZ-L protein). ZZ-BNC acts successfully as a scaffold, enhancing both the sensitivity and binding capacity of IgG, a Fc-fused receptor, and Fc-fused lectin to antigens, cytokines, and sugar chains through an oriented immobilization on a biosensor surface. To expand the versatility of ZZ-BNC, we modified ZZ-BNC by replacing the ZZ domain with a DNA-binding single-chain lambda Cro (scCro) domain, thereby developing scCro-BNC. The scCro-BNC was synthesized in yeast cells and homogeneously purified as ∼30 nm sized nanoparticles. In a quartz crystal microbalance, an scCro-BNC-coated sensor chip immobilized with thrombin-binding DNA aptamers showed an ∼5.5-fold higher thrombin-binding capacity and ∼6000-fold higher detection sensitivity than a sensor chip directly coated with DNA aptamers. In addition, the number of bound thrombin molecules per molecule of DNA aptamer increased by ∼7.8-fold with an scCro-BNC coating, consistent with the theoretical thrombin-binding capacity. Collectively, scCro-BNC was shown to perform as an ideal scaffold for maximizing the potential of the DNA aptamer by immobilizing it in an oriented manner. Facilitating a highly sensitive detection of various target molecules, these BNC-based scaffolds are expected to improve a wide range of biosensors while minimizing the number of sensing molecules required.

Polymerized Albumin Receptor of Hepatitis B Virus for Evading the Reticuloendothelial System.

Various strategies, such as optimization of surface chemistry, size, shape, and charge, have been undertaken to develop nanoparticles (NPs) as DDS (drug delivery system) nanocarriers for evading the reticuloendothelial system (RES) in vivo. We previously developed a hollow NP composed of hepatitis B virus (HBV) surface antigen L proteins and lipid bilayers, hereinafter referred to as bio-nanocapsule (BNC), as a nonviral DDS nanocarrier. Such a BNC harbors the HBV-derived human hepatic cell-specific infection mechanism, and intravenously injected BNCs by themselves were shown to avoid clearance by RES-rich organs and accumulate in target tissues. In this study, since the surface modification with albumins is known to prolong the circulation time of nanomedicines, we examined whether the polymerized albumin receptor (PAR) of BNCs contributes to RES evasion in mouse liver. Our results show that NPs conjugated with peptides possessing sufficient PAR activity were captured by Kupffer cells less efficiently in vitro and were able to circulate for a longer period of time in vivo. Comparing with polyethylene glycol, PAR peptides were shown to reduce the recognition by RES to equal content. Taken together, our results strongly suggest that the PAR domain of BNCs, as well as HBV, harbors an innate RES evasion mechanism. Therefore, the surface modification with PAR peptides could be an alternative strategy for improving the pharmacodynamics and pharmacokinetics of forthcoming nanomedicines.

Influence of Nivolumab for Intercellular Adhesion Force between a T Cell and a Cancer Cell Evaluated by AFM Force Spectroscopy.

The influence of nivolumab on intercellular adhesion forces between T cells and cancer cells was evaluated quantitatively using atomic force microscopy (AFM). Two model T cells, one expressing high levels of programmed cell death protein 1 (PD-1) (PD-1high Jurkat) and the other with low PD-1 expression levels (PD-1low Jurkat), were analyzed. In addition, two model cancer cells, one expressing programmed death-ligand 1 (PD-L1) on the cell surface (PC-9, PD-L1+) and the other without PD-L1 (MCF-7, PD-L1-), were also used. A T cell was attached to the apex of the AFM cantilever using a cup-attached AFM chip, and the intercellular adhesion forces were measured. Although PD-1high T cells adhered strongly to PD-L1+ cancer cells, the adhesion force was smaller than that with PD-L1- cancer cells. After the treatment of PD-1high T cells with nivolumab, the adhesion force with PD-L1+ cancer cells increased to a similar level as with PD-L1- cancer cells. These results can be explained by nivolumab influencing the upregulation of the adhesion ability of PD-1high T cells with PD-L1+ cancer cells. These results were obtained by measuring intercellular adhesion forces quantitatively, indicating the usefulness of single-cell AFM analysis.

Development of a universal method for the measurement of binding affinities of antibody drugs towards a living cell based on AFM force spectroscopy.

A universal method to measure the binding affinities of antibody drugs towards their targets on the surface of living cells was developed based on atomic force microscopy (AFM) analysis. Nivolumab, an antibody drug targeting programmed cell death 1 (PD-1), was mainly used as a model for this evaluation. The surface of a tip-less AFM cantilever was coated with nano-capsules, on which immunoglobulin G-binding ZZ domains of protein A were exposed, and nivolumab molecules were immobilized on the cantilever through binding between the antibody Fc domains and the ZZ domains, which controlled the molecular orientation of the antibodies. Model human T lymphocytes (Jurkat), on which PD-1 molecules were highly expressed, were immobilized on a glass substrate via a lipid bilayer-anchoring reagent. The nivolumab-coated AFM cantilever was moved to approach the T cells, and the rupture forces between nivolumab molecules on the AFM cantilever and PD-1 molecules on the cell surface were measured. The average values of the rupture forces were 0.18 ± 0.10, 0.21 ± 0.18, 0.12 ± 0.07, 0.11 ± 0.06, and 0.12 ± 0.06 nN µm-2 at loading forces of 10, 20, 30, 40, and 50 nN, respectively. Application of significantly higher loading forces decreased the S/N ratio, as confirmed by comparison with control T cells with low PD-1 expression, which suggested that a low loading force of less than 20 nN was sufficient for these measurements. A correlation between the expression levels of PD-1 and the rupture force values was confirmed using immunofluorescence. A similar assay was performed by using an antibody drug targeting epidermal growth factor receptor (EGFR) and a model cancer cell expressing EGFR molecules (A431) to evaluate the universal application of the developed method for various antibody drugs, and the same conclusions as that in nivolumab's case were obtained. This method can be applied to living cells without any chemical treatment, which allows the present method to compare the affinities of various antibody drugs towards the same single cell. These results indicated that the present method is useful for selecting the most effective candidates from various antibody drugs from the point of view of binding forces between antibodies and living cells.

A Novel Hybrid Drug Delivery System for Treatment of Aortic Aneurysms.

Ongoing aortic wall degeneration and subsequent aneurysm exclusion failure are major concerns after an endovascular aneurysm repair with a stent-graft. An ideal solution would be a drug therapy that targets the aortic wall and inhibits wall degeneration. Here, we described a novel drug delivery system, which allowed repetitively charging a graft with therapeutic drugs and releasing them to the aortic wall in vivo. The system was composed of a targeted graft, which was labeled with a small target molecule, and the target-recognizing nanocarrier, which contained suitable drugs. We developed the targeted graft by decorating a biotinylated polyester graft with neutravidin. We created the target-recognizing nanocarrier by conjugating drug-containing liposomes with biotinylated bio-nanocapsules. We successfully demonstrated that the target-recognizing nanocarriers could bind to the targeted graft, both in vitro and in blood vessels of live mice. Moreover, the drug released from our drug delivery system reduced the expression of matrix metalloproteinase-9 in mouse aortas. Thus, this hybrid system represents a first step toward an adjuvant therapy that might improve the long-term outcome of endovascular aneurysm repair.

Enhanced sugar chain detection by oriented immobilization of Fc-fused lectins.

We report a novel scaffold for clustering and oriented immobilization of human IgG1 Fc-fused lectins on biosensors without chemical modifications. This approach uses a bio-nanocapsule (BNC) displaying a tandem form of IgG Fc-binding Z domains derived from Staphylococcus aureus protein A (ZZ-BNC). Incorporating ZZ-BNC effectively increased both the sensitivity and sugar chain-binding capacity compared with the condition without ZZ-BNC.

Two-dimensional membrane scaffold for the oriented immobilization of biosensing molecules.

The orientation and density of biosensing molecules on sensor chip should be precisely controlled to improve sensitivity and ligand-binding capacity. We previously developed a ~30-nm bio-nanocapsule (ZZ-BNC), consisting of the hepatitis B virus envelope L protein fused with the tandem form of protein A-derived IgG Fc-binding Z domain (ZZ-L protein). This is used as a robust nanoparticle scaffold to enhance the sensitivity and ligand-binding capacity of IgGs and Fc-fused sensing molecules (Fc-fused receptors). However, due to their rigid particle structure, the surface density of ZZ-L proteins could not be optimized for biosensor functions, and useless ZZ-L proteins become stuck between ZZ-BNC and the sensor chip. Here, we have developed a planar lipid membrane embedded with ZZ-L micelles (ZZ-L membrane), which could modify the surface of any biosensor chip with a controlled density of ZZ-L proteins. Compared with ZZ-BNC, the sensitivity and ligand-binding capacity of IgGs were enhanced about 10-fold with the ZZ-L membrane. Furthermore, the immobilized IgGs could capture their respective antigens almost stoichiometrically, indicating that ZZ-L membrane is the most ideal scaffold for Fc-fused sensing molecules in terms of both clustering and oriented immobilization.

In vivo uterine local gene delivery system using TAT-displaying bionanocapsules.

BACKGROUND: The uterus is an organ that is directly accessible via the transvaginal route, whereas the drug delivery system and the gene delivery system (GDS) for the uterus are very limited, even in animal models. In the present study, we optimized a bionanocapsule (BNC) comprising a hepatitis B virus envelope L-protein particle, for which a structurally similar particle has been used as an immunogen of a conventional HB vaccine worldwide for more than 30 years, as a local uterine GDS using a mouse model. METHODS: To display various antibodies for re-targeting to different cells other than hepatic cells, the pre-S1 region of BNC was replaced with a tandem form of the protein A-derived immunoglobulin G Fc-interacting region (Z domain, ZZ-BNC). To induce strong cell adhesion after local administration into the uterine cavity, ZZ-BNC was modified with a transactivator of transcription (TAT) peptide. RESULTS: Gene transfer using TAT-modified ZZ-BNC is approximately 5000- or 18-fold more efficient than the introduction of the same dose of naked DNAs or the use of the cationic liposomes, respectively. TAT-modified ZZ-BNC was rapidly eliminated from the uterus and had no effect on the pregnancy rate, litter size or fetal growth. CONCLUSIONS: TAT-modified ZZ-BNC could be a useful GDS for uterine endometrial therapy via local uterine injection.

The Structural Function of Nestin in Cell Body Softening is Correlated with Cancer Cell Metastasis.

Intermediate filaments play significant roles in governing cell stiffness and invasive ability. Nestin is a type VI intermediate filament protein that is highly expressed in several high-metastatic cancer cells. Although inhibition of nestin expression was shown to reduce the metastatic capacity of tumor cells, the relationship between this protein and the mechanism of cancer cell metastasis remains unclear. Here, we show that nestin softens the cell body of the highly metastatic mouse breast cancer cell line FP10SC2, thereby enhancing the metastasis capacity. Proximity ligation assay demonstrated increased binding between actin and vimentin in nestin knockout cells. Because nestin copolymerizes with vimentin and nestin has an extremely long tail domain in its C-terminal region, we hypothesized that the tail domain functions as a steric inhibitor of the vimentin-actin interaction and suppresses association of vimentin filaments with the cortical actin cytoskeleton, leading to reduced cell stiffness. To demonstrate this function, we mechanically pulled vimentin filaments in living cells using a nanoneedle modified with vimentin-specific antibodies under manipulation by atomic force microscopy (AFM). The tensile test revealed that mobility of vimentin filaments was increased by nestin expression in FP10SC2 cells.

Oriented immobilization to nanoparticles enhanced the therapeutic efficacy of antibody drugs.

Antibody drugs have been important therapeutic agents for treating various diseases, such as cancer, rheumatism, and hypercholesterolemia, for the last three decades. Despite showing excellent therapeutic efficacy with good safety in vivo, they require high doses. We have developed a ∼30-nm bio-nanocapsule (ZZ-BNC) consisting of hepatitis B virus envelope L protein fused with the tandem form of protein A-derived IgG Fc-binding Z domain (ZZ-L protein), for tethering antibodies in an oriented immobilization manner. In this study, antibody drugs were spontaneously conjugated to ZZ-BNC, which displayed the IgG Fv regions outwardly. The anti-human epidermal growth factor receptor IgG conjugated to ZZ-BNC (α-hEGFR-ZZ-BNC) was endocytosed by the human epidermoid carcinoma A431 cells, with increases in cellular uptake by ∼1.5 fold, compared that of α-hEGFR IgG alone. The amount of α-hEGFR IgG in the late endosomes and lysosomes was increased from 4% to 33% by the conjugation to ZZ-BNC. The in vitro cytotoxicity of α-hEGFR-ZZ-BNC was higher by ∼10-fold than that of α-hEGFR IgG alone. Furthermore, in vivo tumor growth was significantly reduced by α-hEGFR-ZZ-BNC than by α-hEGFR IgG alone. Taken together, since endosomal EGFR, not cell surface EGFR, played a pivotal role in the EGFR-mediated signaling cascade, ZZ-BNC increased α-hEGFR IgG avidity by efficiently repressing the activation of hEGFR not only on the cell surface, but presumably also in the endosomes. These results strongly suggested that ZZ-BNC is a promising nano-scaffold for enhancing the therapeutic efficacy and reducing the dose of antibody drugs. STATEMENT OF SIGNIFICANCE: Antibody drugs are widely used for treating severe diseases, such as cancer, rheumatism, and hypercholesterolemia. These drugs are composed of naturally occurring biomaterials with low immunogenicity and toxicity, as well as long in vivo serum half-life. To achieve sufficient therapeutic efficacy, the dose of antibody drugs are unavoidably higher than those of conventional drugs. The present study shows an innovative way to reduce the dose of antibody drugs by using a nanocarrier-conjugated antibody. Oriented immobilization of the antibody enhanced its avidity, endocytosis efficiency, and therapeutic efficacy.

A hepatitis B virus-derived human hepatic cell-specific heparin-binding peptide: identification and application to a drug delivery system.

Viruses are naturally evolved nanocarriers that can evade host immune systems, attach specifically to the surfaces of target cells, enter the cells through endocytosis, escape from endosomes efficiently, and then transfer their genomes to host cells. Hepatitis B virus (HBV) is a ∼42 nm enveloped DNA virus that can specifically infect human hepatic cells. To utilize the HBV-derived early infection machinery in synthetic nanocarriers, the human hepatic cell-binding site (i.e., the sodium taurocholate co-transporting polypeptide (NTCP)-binding site, with myristoylated pre-S1(2-47)) and the low pH-dependent fusogenic domain (pre-S1(9-24)) are indispensable for targeting and endosomal escape, respectively. However, cell-surface NTCP has recently been shown not to be involved in the initial attachment of HBV. In this study, we identified a novel heparin-binding site (pre-S1(30-42)) in the N-terminal half of the pre-S1 region, which presumably interacts with cell-surface heparan sulfate proteoglycan (HSPG) and plays a pivotal role in the initial attachment of HBV to human hepatic cells. The evolutionarily conserved amino acid residues Asp-31, Trp-32, and Asp-33 are indispensable for the heparin-binding activity. Liposomes (LPs) displaying the peptide were endocytosed by human hepatic cells in a cell-surface heparin-dependent manner and delivered doxorubicin to human hepatic cells more efficiently than myristoylated pre-S1(2-47)-displaying LPs. These results demonstrated that the pre-S1(30-42) peptide is the most promising HBV-derived targeting peptide for synthetic nanocarriers, and that this peptide exhibits high specificity for human hepatic cells and efficiently induces endocytosis.

CD11c-specific bio-nanocapsule enhances vaccine immunogenicity by targeting immune cells.

BACKGROUND: Various nanocarriers have been used to deliver subunit vaccines specifically to dendritic cells (DCs) for the improvement of immunogenicity. However, due to their insufficient DC priming ability, these vaccines could not elicit effective innate immunity. We have recently developed a DC-targeting bio-nanocapsule (BNC) by displaying anti-CD11c IgGs via protein A-derived IgG Fc-binding Z domain on the hepatitis B virus envelope L protein particles (α-DC-ZZ-BNC). RESULTS: After the chemical modification with antigens (Ags), the α-DC-ZZ-BNC-Ag complex could deliver Ags to DCs efficiently, leading to effective DC maturation and efficient endosomal escape of Ags, followed by Ag-specific T cell responses and IgG productions. Moreover, the α-DC-ZZ-BNC modified with Japanese encephalitis virus (JEV) envelope-derived D3 Ags could confer protection against 50-fold lethal dose of JEV injection on mice. CONCLUSION: The α-DC-ZZ-BNC-Ag platform was shown to induce humoral and cellular immunities effectively without any adjuvant.

Development of a macrophage-targeting and phagocytosis-inducing bio-nanocapsule-based nanocarrier for drug delivery.

Macrophage hyperfunction or dysfunction is tightly associated with various diseases, such as osteoporosis, inflammatory disorder, and cancers. However, nearly all conventional drug delivery system (DDS) nanocarriers utilize endocytosis for entering target cells; thus, the development of macrophage-targeting and phagocytosis-inducing DDS nanocarriers for treating these diseases is required. In this study, we developed a hepatitis B virus (HBV) envelope L particle (i.e., bio-nanocapsule (BNC)) outwardly displaying a tandem form of protein G-derived IgG Fc-binding domain and protein L-derived IgG Fab-binding domain (GL-BNC). When conjugated with the macrophage-targeting ligand, mouse IgG2a (mIgG2a), the GL-BNC itself, and the liposome-fused GL-BNC (i.e., GL-virosome) spontaneously initiated aggregation by bridging between the Fc-binding domain and Fab-binding domain with mIgG2a. The aggregates were efficiently taken up by macrophages, whereas this was inhibited by latrunculin B, a phagocytosis-specific inhibitor. The mIgG2a-GL-virosome containing doxorubicin exhibited higher cytotoxicity toward macrophages than conventional liposomes and other BNC-based virosomes. Thus, GL-BNCs and GL-virosomes may constitute promising macrophage-targeting and phagocytosis-inducing DDS nanocarriers. STATEMENT OF SIGNIFICANCE: We have developed a novel macrophage-targeting and phagocytosis-inducing bio-nanocapsule (BNC)-based nanocarrier named GL-BNC, which comprises a hepatitis B virus envelope L particle outwardly displaying protein G-derived IgG Fc- and protein L-derived IgG Fab-binding domains in tandem. The GL-BNC alone or liposome-fused form (GL-virosomes) could spontaneously aggregate when conjugated with macrophage-targeting IgGs, inducing phagocytosis by the interaction between IgG Fc of aggregates and FcγR on phagocytes. Thereby these aggregates were efficiently taken up by macrophages. GL-virosomes containing doxorubicin exhibited higher cytotoxicity towards macrophages than ZZ-virosomes and liposomes. Our results suggested that GL-BNCs and GL-virosomes would serve as promising drug delivery system nanocarriers for targeting delivery to macrophages.

A New Cell Separation Method Based on Antibody-Immobilized Nanoneedle Arrays for the Detection of Intracellular Markers.

Focusing on intracellular targets, we propose a new cell separation technique based on a nanoneedle array (NNA) device, which allows simultaneous insertion of multiple needles into multiple cells. The device is designed to target and lift ("fish") individual cells from a mixed population of cells on a substrate using an antibody-functionalized NNA. The mechanics underlying this approach were validated by force analysis using an atomic force microscope. Accurate high-throughput separation was achieved using one-to-one contacts between the nanoneedles and the cells by preparing a single-cell array in which the positions of the cells were aligned with 10,000 nanoneedles in the NNA. Cell-type-specific separation was realized by controlling the adhesion force so that the cells could be detached in cell-type-independent manner. Separation of nestin-expressing neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) was demonstrated using the proposed technology, and successful differentiation to neuronal cells was confirmed.

Synthesis and assembly of Hepatitis B virus envelope protein-derived particles in Escherichia coli.

Hepatitis B virus (HBV) envelope particles have been synthesized in eukaryotic cells (e.g., mammalian cells, insect cells, and yeast cells) as an HB vaccine immunogen and drug delivery system (DDS) nanocarrier. Many researchers had made attempts to synthesize the particles in Escherichia coli for minimize the cost and time for producing HBV envelope particles, but the protein was too deleterious to be synthesized in E. coli. In this study, we generated deletion mutants of HBV envelope L protein (389 amino acid residues (aa)) containing three transmembrane domains (TM1, TM2, TM3). The ΔNC mutant spanning from TM2 to N-terminal half of TM3 (from 237 aa to 335 aa) was found as a shortest form showing spontaneous particle formation. After the N-terminal end of ΔNC mutant was optimized by the N-end rule for E. coli expression, the modified ΔNC mutant (mΔNC) was efficiently expressed as particles in E. coli. The molecular mass of mΔNC particle was approx. 670 kDa, and the diameter was 28.5 ± 6.2 nm (mean ± SD, N = 61). The particle could react with anti-HBV envelope S protein antibody, indicating the particles exhibited S antigenic domain outside as well as HBV envelope particles. Taken together, the E. coli-derived mΔNC particles could be used as a substitute of eukaryotic cell-derived HBV envelope particles for versatile applications.

Scaffolds for oriented and close-packed immobilization of immunoglobulins.

Immunosensing is a widely used technique that detects the interactions between antibodies and antigens such as biochemical markers, pathogens, allergens, and tumor-associated antigens. Since target antigens are often of high molecular mass and their binding affinities are sometimes weak, the spatial arrangement of immunoglobulin Gs (IgGs) on immunosensing probes should be optimized by presenting them in as close-packed a manner as possible and reducing steric hindrance around the antigen-binding Fv regions. Both clustering and oriented immobilization of IgGs on immunosensing probes are thus important for enhancing the sensitivity and antigen-binding capacity of probes. Intact IgGs, IgG-derived fragments, or IgG-compatible fragments have previously been clustered onto solid phases with a variety of scaffold chemistries (e.g., crosslinkers, polymers, self-assembled monolayers, protein A/G, avidin, DNA) to improve immunosensing probes, none of these strategies has yet accomplished both clustering and oriented immobilization of IgGs. Recently, we developed an ~30-nm bio-nanocapsule (ZZ-BNC), consisting of transmembrane ZZ-L protein deploying a tandem form of the IgG Fc-binding Z domain derived from Staphylococcus aureus protein A on its outer surface that functioned as a scaffold for the clustering and oriented immobilization of IgGs and Fc-fused biosensing molecules. In this review, we present an overview of conventional techniques for IgG immobilization and describe the molecular basis of the ZZ-BNC-based technology, discussing the potential and versatility of this technology not only in immunosensors but also in other types of biosensors.