Cost-effective production of tag-less recombinant protein in Nicotiana benthamiana.

Plants have recently received a great deal of attention as a means of producing recombinant proteins. Despite this, a limited number of recombinant proteins are currently on the market and, if plants are to be more widely used, a cost-effective and efficient purification method is urgently needed. Although affinity tags are convenient tools for protein purification, the presence of a tag on the recombinant protein is undesirable for many applications. A cost-effective method of purification using an affinity tag and the removal of the tag after purification has been developed. The family 3 cellulose-binding domain (CBM3), which binds to microcrystalline cellulose, served as the affinity tag and the small ubiquitin-related modifier (SUMO) and SUMO-specific protease were used to remove it. This method, together with size-exclusion chromatography, enabled purification of human interleukin-6 (hIL6) with a yield of 18.49 mg/kg fresh weight from leaf extracts of Nicotiana benthamiana following Agrobacterium-mediated transient expression. Plant-produced hIL6 (P-hIL6) contained less than 0.2 EU/µg (0.02 ng/mL) endotoxin. P-hIL6 activated the Janus kinase-signal transducer and activator of transcriptional pathways in human LNCaP cells, and induced expression of IL-21 in activated mouse CD4+ T cells. This approach is thus a powerful method for producing recombinant proteins in plants.

Nanoscale analysis of a functionalized polythiophene surface by adhesion mapping.

Functionalized ethylenedioxythiophene (EDOT) monomers, hydroxymethyl EDOT (EDOT-OH), and zwitterionic phosphorylcholine EDOT (EDOT-PC) were electropolymerized to prepare the homopolymers poly(EDOT-OH) and poly(EDOT-PC), and mixtures of these monomers were used to produce the copolymer poly(EDOT-OH)-co-poly(EDOT-PC). Force-extension-curve-based atomic force microscopy (AFM) was utilized to analyze the surfaces of the films. The PEDOT-OH film yielded force-extension curves for short stretching, and the PEDOT-PC film yielded curves for long stretching. A dendron-modified AFM tip with anthracene groups tethered at the end resulted in adhesion maps with the highest contrast. The analytical data for the copolymer films correlated with the corresponding monomer composition, and the maps revealed that the average size for the copolymer nanodomains ranged from 10-14 nm. This approach can be applied to studies aimed at understanding the surface structure of other relevant polymers and copolymers at the nanoscale level.

Phosphokinase antibody arrays on dendron-coated surface.

Monitoring protein phosphorylation at the cellular level is important to understand the intracellular signaling. Among the phosphoproteomics methods, phosphokinase antibody arrays have emerged as preferred tools to measure well-characterized phosphorylation in the intracellular signaling. Here, we present a dendron-coated phosphokinase antibody array (DPA) in which the antibodies are immobilized on a dendron-coated glass slide. Self-assembly of conically shaped dendrons well-controlled in size and structure resulted in precisely controlled lateral spacing between the immobilized phosphosite-specific antibodies, leading to minimized steric hindrance and improved antigen-antibody binding kinetics. These features increased sensitivity, selectivity, and reproducibility in measured amounts of protein phosphorylation. To demonstrate the utility of the DPA, we generated the phosphorylation profiles of brain tissue samples obtained from Alzheimer's disease (AD) model mice. The analysis of the profiles revealed signaling pathways deregulated during the course of AD progression.

Direct quantitative analysis of HCV RNA by atomic force microscopy without labeling or amplification.

Force-based atomic force microscopy (AFM) was used to detect HCV (hepatitis C virus) RNA directly and to quantitatively analyse it without the need for reverse transcription or amplification. Capture and detection DNA probes were designed. The former was spotted onto a substrate with a conventional microarrayer, and the latter was immobilized on an AFM probe. To control the spacing between the immobilized DNAs on the surface, dendron self-assembly was employed. Force-distance curves showed that the mean force of the specific unbinding events was 32 ± 5 pN, and the hydrodynamic distance of the captured RNA was 30-60 nm. Adhesion force maps were generated with criteria including the mean force value, probability of obtaining the specific curves and hydrodynamic distance. The maps for the samples whose concentrations ranged from 0.76 fM to 6.0 fM showed that cluster number has a linear relationship with RNA concentration, while the difference between the observed number and the calculated one increased at low concentrations. Because the detection limit is expected to be enhanced by a factor of 10 000 when a spot of 1 micron diameter is employed, it is believed that HCV RNA of a few copy numbers can be detected by the use of AFM.

"Seeing and counting" individual antigens captured on a microarrayed spot with force-based atomic force microscopy.

The mapping capability of atomic force microscopy (AFM) enabled us to see captured prostate-specific antigens (PSAs) on a spot microarrayed with the corresponding antibody and count the number of the antigens in a submicrometer area. To enhance the reliability and the reproducibility of the approach, a third-generation dendron was employed for the surface treatment. The specific force between the captured PSA and the detection antibody (5A6) was measured after cross-linking, and the mean unbinding force was 56 +/- 2 pN. At 100 fM, there were 12 captured antigens in 4.32 x 10(4) nm(2), and the number was dependent upon the concentration. A larger hydrodynamic distance (8 +/- 2 nm) of the immunocomplex resulted in a cluster of pixels corresponding to the single complex in a map recorded over a selected area with a positional interval of 3 nm, and this feature helped to discriminate between pixels of the specific interaction and the nonspecific ones. The results indicate that the approach can be applicable to the quantitative analysis of the antigen in a sample and imply that it can be extended to a sample of very low copy numbers as long as the size of the microarrayed spot is reduced.

Nanotechnology for early cancer detection.

Vast numbers of studies and developments in the nanotechnology area have been conducted and many nanomaterials have been utilized to detect cancers at early stages. Nanomaterials have unique physical, optical and electrical properties that have proven to be very useful in sensing. Quantum dots, gold nanoparticles, magnetic nanoparticles, carbon nanotubes, gold nanowires and many other materials have been developed over the years, alongside the discovery of a wide range of biomarkers to lower the detection limit of cancer biomarkers. Proteins, antibody fragments, DNA fragments, and RNA fragments are the base of cancer biomarkers and have been used as targets in cancer detection and monitoring. It is highly anticipated that in the near future, we might be able to detect cancer at a very early stage, providing a much higher chance of treatment.

Dendron-modified polystyrene microtiter plate: surface characterization with picoforce AFM and influence of spacing between immobilized amyloid beta proteins.

A polystyrene microtiter plate was coated with a molecular layer of a cone-shaped dendron as a means of providing proper spacing between immobilized biomolecules. For the coating preparation, di(ethylene glycol) vinyl ether was grafted onto the surface of the microtiter plate by a plasma process followed by self-assembly of a second-generation dendron (9-acid) or a third-generation dendron (27-acid). Contact angle analysis revealed a pronounced increase in the hydrophilicity upon plasma grafting, while the hydrophilicity reverted/decreased after dendron immobilization. For analysis by force-based atomic force microscopy (AFM), oligonucleotides were immobilized onto the AFM tip and the plate. The DNA-DNA interaction was observed at all spots examined, which implied that coating of the dendrons was uniform over the entire surface. The effectiveness for biomolecular assays of the spacing on dendron-modified microtiter plates was examined by carrying out an enzyme-linked immunosorbent assay (ELISA), where enhanced detection of different fragments of amyloid beta protein (A beta) was observed when compared with other conventional plates, such as untreated polystyrene or maleic anhydride activated plates. The positive influence of the mesospacing between biomolecules on the microtiter plates for this assay was confirmed.

Potential lead for an Alzheimer drug: a peptide that blocks intermolecular interaction and amyloid beta protein-induced cytotoxicity.

A peptide chAbeta30-16 (15-mer; CTFVRTHIFCKEHQF) was designed to bind to a region encompassing the entire polymerization-related (16KLVFF20) and part of the polymerization and toxicity-related (25GSNKGAIIGLM35) regions of amyloid beta-protein, Abeta1-42 by a hydropathic complementary approach. This peptide efficiently binds to Abeta and blocks intermolecular interaction and the formation of Abeta aggregates. In addition, the peptide neutralizes the cell toxicity of Abeta fibrils. The chAbeta30-16 peptide or its derivatives may be a starting point for the future development of drugs that prevent the neurotoxicity and deposition of Abeta in the brain of Alzheimer's disease.

Expression and functional reconstitution of a recombinant antibody (Fab') specific for human apolipoprotein B-100.

We have cloned and constructed plasmid vectors, pETB23H and pETB23L, for bacterial expression of heavy (H) and light (L) chain cDNAs of Fab' of mAbB23 a monoclonal antibody specific to human plasma apolipoprotein (apo) B-100. The H- and L-chains were expressed as insoluble inclusion bodies in the cytoplasm of Escherichia coli. The inclusion bodies of both chains were isolated from the cell lysate, solubilized in 6 M guanidium-HCl, and mixed in equal molar amounts. Refolding was performed in three stages of dialysis: first, dialysis against 3 M guanidium buffer, next, continuous decrement of guanidium in the dialysis buffer through slow addition of 1 M guanidium buffer, and finally, dialysis against a buffer without guanidium. After the refolding, active Fab' (rFab') was purified through an apo B-100-coupled affinity column. When compared by ELISA, the rFab' had a slightly decreased antigen-binding activity (about 0.7-fold) compared with native Fab. The refolding yield was maximum (75%) when performed at the protein concentrations not more than 0.4 mg ml(-1), whereas the yield decreased exponentially at higher concentrations. The maximum recovery was obtained at the refolding concentration of 1.8 mg ml(-1), where the yield was about 45%. Overall, 2.4-3.0 mg of active rFab' specific to apo B-100 was successfully obtained from 1 l cultivation of E. coli cells.

Bacterial expression and in vitro refolding of a single-chain fv antibody specific for human plasma apolipoprotein B-100.

From the cloned heavy and light chains of a murine monoclonal antibody (mAbB23) which is specific for human apolipoprotein (apo) B-100 of plasma low-density lipoproteins, a vector was designed for expression of a single-chain antibody (scFv) of mAbB23 in Escherichia coli. The expression vector was constructed so that the scFv gene (V(L)-linker-V(H)) was expressed under the control of the T7 promoter. The inclusion body of scFv was isolated from E. coli lysate and solubilized in 6 M guanidine-hydrochloride without reducing agents, followed by refolding through slow dilution into refolding buffer. After complete removal of the remaining denaturant by dialysis, the soluble scFv was purified through an apo B-100-coupled affinity column, and an active fraction, which had an antigen-binding activity comparable with that of native Fab, was easily obtained. The expression and in vitro refolding of scFv resulted in production of an active molecule in a yield of 15-20 mg per 1-liter flask cultivation.