Production of influenza virus-like particles using recombinant insect cells.

Virus-like particles (VLPs) are hollow nanoparticles composed of recombinant viral surface proteins without a virus genome. In the present study, we investigated the production of influenza VLPs using recombinant insect cells. DNA fragments encoding influenza A virus hemagglutinin (HA) and matrix protein 1 (M1) were cloned with the Drosophila BiP signal sequence in plasmid vectors containing a blasticidin and a neomycin resistance gene, respectively. After Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with a pair of constructed plasmid vectors, stably transformed cells were established via incubation with blasticidin and G418. Western blot analyses showed that recombinant High Five cells secreted HA and M1 proteins into the culture supernatant. Immunoprecipitation of the culture supernatant with an anti-HA antibody and transmission electron microscopy suggested that secreted HA and M1 proteins were in a particulate structure with a morphology similar to that of an influenza virus. Hemagglutination assay indicated that expressed HA molecules retained hemagglutination activity. In a shake-flask culture, recombinant cells achieved a high HA yield (≈ 10 µg/ml) comparable to the yields obtained using the baculovirus-insect cell system. Recombinant insect cells may serve as excellent platforms for the efficient production of influenza VLPs for use as safe and effective vaccines and diagnostic antigens.

Efficient production of Japanese encephalitis virus-like particles by recombinant lepidopteran insect cells.

The production of Japanese encephalitis (JE) virus-like particles (VLPs) in stably transformed lepidopteran insect cells was investigated. The DNA fragment encoding the JE virus (JEV) prM signal peptide, the precursor (prM) of the viral membrane protein (M), and the envelope glycoprotein (E) was cloned into the plasmid vector pIHAbla. The pIHAbla contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression, together with a blasticidin resistance gene for use as a selectable marker. DNA encoding a form of prM with a pr/M cleavage site mutation was used to suppress the cell-fusion activity of VLPs. After transfection with the resultant plasmid, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were incubated with blasticidin, and cells resistant to the antibiotic were obtained. Western blot analysis and enzyme-linked immunosorbent assay of a culture supernatant showed that transfected High Five cells secreted an E antigen equivalent to the authentic JEV E. Sucrose density-gradient sedimentation analysis of the culture supernatant from recombinant High Five cells indicated that secreted E antigen molecules were produced in a particulate form. VLPs recovered from the supernatant successfully induced neutralizing antibodies in mice, particularly when adsorbed to alum adjuvant. High yields (≈30 µg/ml) of E antigen were achieved in shake-flask cultures. These results indicate that recombinant insect cells may offer a novel approach for efficient VLP production.

Effects of fluid-flow regimes on Chlorella sorokiniana cultivation in cascade photobioreactors with either flat or wavy bottoms.

Computational fluid dynamics (CFD) was used to investigate cascade photobioreactors (cascade PBRs) with two different bottom configurations-flat and wavy-to establish the effect that fluid-flow regimes exert on the photosynthetic productivity of Chlorella sorokiniana. In the flat-bottom PBR, areal biomass productivities decreased from 6.8 to 4.2 g·m-2·d-1 when the flow rate of a culture per unit of lane width was increased from 33 to 132 L·m-1·min-1. We found that this decrease in the areal productivity was the result of a decrease in the volumetric photon flux densities (volumetric PFDs), which was caused by an increase in the depth of the culture in the lane. Through CFD calculation and long-exposure photography, the flow of the culture in the wavy-bottom PBR was characterized in an upper straightforward section and underneath the swirling section. Under identical conditions of flow rate and volumetric PFD (66 L·m-1·min-1 and 50 µmol·m-3·s-1, respectively), the cell growth accelerated in the wavy-bottom PBR with areal productivity that reached 6.5 g·m-2·d-1-productivity was 5.1 g·m-2·d-1 in the flat-bottom PBR. The swirling flow in the wave troughs held the culture for longer periods in the illuminated lane, and the resultant extended period of mixing improved the photosynthetic productivity.

Functional expression of single-chain Fv antibody in the cytoplasm of Escherichia coli by thioredoxin fusion and co-expression of molecular chaperones.

The production of a single-chain variable fragment (scFv) antibody against bovine ribonuclease A in the cytoplasm of Escherichia coli trxB/gor double mutant was investigated. Previous reports have shown that the thioredoxin (Trx) protein fusion strategy is useful for the correct folding of scFvs and that the expression of functional scFvs is increased by co-expression of molecular chaperones. In the present study, we examined the effects of the combination of Trx fusion and molecular chaperone co-expression on the production of a functional scFv. A Trx-fused scFv was obtained in the oxidizing cytoplasm, and co-expression of GroELS and trigger factor had the greatest effect, resulting in a 2.8-fold increase in specific productivity. By contrast, the molecular chaperone DnaKJE had no effect. Moreover, co-expression of DnaKJE with GroELS negated the effects of GroELS. Trx-scFv was purified using a bovine ribonuclease A-coupled Sepharose column, and 2.7 mg/L of purified protein was obtained. Soluble Trx-scFv, expressed and purified as described above, exhibited pH-dependent binding similar to that of the parental full-length antibody. In addition, approximately 80% of the initial binding activity was retained after incubation at 37 degrees C for 2 weeks, indicating that the Trx-scFv fusion protein is quite stable. This strategy might be useful for the preparation of other recombinant scFvs.

Production of an antibody Fab fragment using 2A peptide in insect cells.

Antibody Fab fragments consist of heavy chain (Hc) and light chain (Lc) polypeptides assembled with a disulphide bond. The production of a recombinant Fab fragment requires the simultaneous expression of two genes encoding both an Hc and an Lc in the same host cell. In the present study, we investigated the production of Fab fragments in lepidopteran insect cells using a bicistronic plasmid vector carrying the Hc and Lc genes linked with a 2A self-cleaving peptide sequence from the porcine teschovirus-1. We also examined the arrangement of a GSG spacer sequence and a furin cleavage site sequence with the 2A sequence. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) of culture supernatants showed that Trichoplusia ni BTI-TN-5B1-4 (High Five) cells transfected with a plasmid in which the Hc and Lc genes were joined by the 2A sequence successfully secreted Fab fragments with antigen-binding activity after self-cleavage of the 2A peptide. The GSG linker enhanced 2A cleavage efficiency, and the furin recognition site was useful for removal of 2A residues from the Hc. Transfection with a single plasmid that contained sequences for GSG, the furin cleavage site, GSG, and the 2A peptide between the Hc and Lc genes exhibited a higher productivity than co-transfection with a set of plasmids separately carrying the Hc or Lc gene. These results demonstrate that bicistronic expression with the appropriate combination of a furin recognition site, GSG linkers, and a 2A peptide may be an effective way to efficiently produce recombinant antibody molecules in insect cells.

Effects of autophagy inducers on recombinant antibody production in insect cells.

Insect cells have recently proven to be an excellent platform for the high-level production of functional recombinant proteins. Autophagy is an important mechanism that promotes cell survival by eliminating damaged organelles and protein aggregates, and it also may influence recombinant protein production. In the present study, we compared the effects that autophagy inducers rapamycin, everolimus, and lithium chloride exert on recombinant lepidopteran insect cells that secrete an engineered antibody molecule. Compared with nontreatment, treatment with either rapamycin or everolimus prolonged cell growth to allow high cell density, improved viability in the declining phase, and then increased the yield of secreted antibodies. These positive effects appeared to be induced via autophagy since autophagosomes were clearly detected, particularly in cells treated with rapamycin or everolimus. Unlike rapamycin, another autophagy inducer, FK506, was ineffective in insect cells. The addition of an appropriate autophagy inducer may be effective in increasing the productivity of recombinant proteins in insect cells.

Effects of lithium on the secretory production of recombinant antibody from insect cells.

Monoclonal antibodies and antibody fragments are widely used in therapeutics and diagnoses. While mammalian cells serve as the host cells for antibody production, insect cells can produce large quantities of secretory antibodies in serum-free suspension cultures. The effects of lithium on the processes of autophagy and apoptosis in mammalian cells are well chronicled. In the present study, stably transformed insect cells, which produce an engineered antibody molecule, were cultured with lithium chloride in a serum-free medium. Treatment with lithium chloride induced autophagy and apoptosis in recombinant insect cells and led to increases in the yields of secreted antibodies.

Effect of light intensity and frequency of flashing light from blue light emitting diodes on astaxanthin production by Haematococcus pluvialis.

Flashing light from blue light emitting diodes is an effective method for the reduction of energy consumption in the bioproduction of astaxanthin by Haematococcus pluvialis. We investigated the effects of light intensity and frequency on the final astaxanthin concentration in bioproduction by H. pluvialis grown mixotrophically. The final astaxanthin concentration under illumination with flashing light, with frequencies ranging from 25 to 200 Hz, was dependent on the light intensity and on the duty cycle and was equivalent, or higher, in comparison with that under illumination with continuous light at the same incident intensity. The light intensity determined the maximum attainable concentration of astaxanthin under continuous illumination. Under illumination with flashing light, the ratio of the final astaxanthin concentration to the maximum concentration at a specific light intensity was correlated to the duty cycle in the frequency range from 25 to 200 Hz. The effect of lower frequencies on enhanced astaxanthin production under flashing light was also studied; at levels as low as 1 Hz, higher final astaxanthin concentrations were observed under flashing light compared to concentrations attained under continuous light.

Surface grafting techniques on the improvement of membrane bioreactor: State-of-the-art advances.

Membrane bioreactor (MBR) is regarded as the state-of-the-art technology in separation processes. Surface modification techniques play a critical role in improving the conventional membrane system which is mostly hydrophobic in nature. The hydrophobic nature of membranes is known to cause fouling, resulting in high maintenance costs and shorter lifespan of MBR. Thus, surface grafting aims to improve the hydrophilicity of bio-based membrane systems. This review describes the major surface grafting techniques currently used in membranes, including photo induced grafting, plasma treatment and plasma induced grafting, radiation induced grafting, thermal induced grafting and ozone induced grafting. The advantages and disadvantages of each method is discussed along with their parametric studies. The potential applications of MBR are very promising, but some integral membrane properties could be a major challenge that hinders its wider reach. The fouling issue could be resolved with the surface grafting techniques to achieve better performance of MBRs.

Optimization of silica-based media for antibody purification by protein A affinity chromatography.

Considering the large molecular size of IgG antibodies widely used for therapeutic applications, the pore size, pore volume and coupling density of silica-based media were optimized for the effective large-scale purification, using an antibody-protein A affinity system. Silica media, with average pore sizes from 70 nm to 140 nm and surface areas of 26-67 m(2)/g, were prepared and coupled with protein A. The static adsorption capacity and dynamic binding capacity of bovine and human IgG were measured at superficial liquid velocities ranging from 94 to 720 cm/h. The volumetric coefficient of mass transfer of the alkali-treated silica-based protein A media, with a pore size of 110 nm, was four times higher than the values for cross-linked agarose media and thus had high dynamic binding capacities at high superficial liquid velocities.

Production of single-chain variable fragment antibody (scFv) in fed-batch and continuous culture of Pichia pastoris by two different methanol feeding methods.

This study examined the effects of two methods of methanol feeding, DO-stat and methanol concentration control, in fed-batch and continuous cultures of Pichia pastoris on cell growth and single-chain variable fragment antibody (scFv) expression. By maintaining the methanol concentration at 3.9 g l(-1) in fed-batch culture, a scFv concentration of 198 mg l(-1) was obtained. In continuous culture using both methanol feeding methods, the scFv concentration in the fermentation broth increased with a decreasing dilution rate. A maximum scFv concentration of 810 mg l(-1) at a dilution rate of 0.0094 h(-1) was obtained by maintaining the methanol concentration at 3.9 g l(-1). Although the specific methanol consumption rate was the same for both methods, the specific productivity of scFv was higher in methanol concentration control from 0.0094 to 0.049 h(-1) than it was in DO-stat control. Therefore, continuous culture with methanol feeding by the concentration control method shows promise for the industrial scale production of recombinant proteins by Pichia pastoris.

Efficient production of antibody Fab fragment by transient gene expression in insect cells.

Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins.

Single nucleotide polymorphism detection method by temperature-gradient affinity chromatography using a single-stranded oligo-DNA coupled column.

We developed an affinity chromatographic method for simple single nucleotide polymorphism (SNP) detection by use of a single-stranded DNA-coupled column and temperature gradient elution, utilizing the difference in thermal stability between hybridized double-stranded DNAs with and without mismatched base-pairs in the course of temperature gradient elution. We studied experimentally and theoretically the elution behavior of DNAs with and without SNPs in this chromatography and proposed a numerical calculation method based on a thermodynamic dissociation model. The effects of the column volume, flow rate of eluent and heating rate of the column on elution profiles were clarified. For designing DNA ligands, mismatched base-pair positions favorable for detection of SNPs were also explored by use of hybridized DNAs coding a part of the human TP53 gene.

New detection method for hydrogen gas for screening hydrogen-producing microorganisms using water-soluble wilkinson's catalyst derivative.

A water-soluble color indicator was developed for the effective screening of hydrogen-producing microorganisms. This indicator consists of a coloring agent and a water-soluble derivative of Wilkinson's catalyst. Wilkinson's catalyst, Tris(triphenylphosphine) rhodium chloride, had been developed as a catalyst for the hydrogenation of olefins. We used a sulfonate of the catalyst for the hydrogenation of coloring agent in an aqueous medium. Several coloring agents, such as methyl orange, methyl red sodium, neutral red and Evan's blue, dissolved in water together with the sulfonated catalyst showed a change in color when hydrogen gas was fed into the solution by sparging at room temperature. We confirmed that methyl orange was decolorized by biologically produced hydrogen, when the photosynthetic bacterial strain Rhodobacter capsulatus ST-410 was grown in a medium containing 0.6 mM catalyst and 0.075 mM methyl orange in test tubes of 5 ml working volume.

Effect of flashing light from blue light emitting diodes on cell growth and astaxanthin production of Haematococcus pluvialis.

To conserve energy in the production of astaxanthin by the green alga Haematococcus pluvialis, we utilized intermittent flashing light from blue light emitting diodes (LEDs) and investigated the effects of the incident light intensity (2-12 micromol m(-2) s(-1)), duty cycle (17-67%) and frequency (25-200 Hz) of flashing on the cell growth and astaxanthin production. In the above ranges, the final astaxanthin concentration under illumination by flashing light was significantly higher than that obtained under illumination with continuous light at the same incident intensity. For example, flashing light at an incident intensity of 8 micromol m(-2) s(-1) gave the same final astaxanthin concentration that was obtained under continuous light illumination at 12 micromol m(-2) s(-1), thus reducing energy consumption by 1/3. We therefore conclude that flashing light from blue LEDs is a promising illumination method for indoor algal cultivation using photobioreactors.

Effective utilization of transmitted light for astaxanthin production by Haematococcus pluvialis.

A new cultivation method, in which physiological responses of Haematococcus pluvialis to different intensities and wavelengths of illuminating light are utilized, is proposed. In this method, light transmitted through a cultivation vessel illuminated from the opposite side was utilized for the early-phase cultivation of H. pluvialis in another inoculated vessel that was located behind the cultivation vessel, to save time required for the growth of cells. After harvesting cells from the front vessel, the vessel that was originally behind was shifted to the position of the front vessel. The abrupt increase in light intensity caused by shift of the position induced the effective accumulation of astaxanthin. These procedures for inoculation, shift of vessels and harvest were repeated using two vessels arranged in series along the light path. After four repeated cycles of cultivation (840 h from the start of cultivation), using 2.5 cm thick vessels, astaxanthin production per unit volume and the astaxanthin content were higher than obtained in a batch cultivation with two 2.5 cm vessels.

Degradation of cyanuric acid in soil by Pseudomonas sp. NRRL B-12227 using bioremediation with self-immobilization system.

The rates of degradation of cyanuric acid, a key intermediate in a metabolic pathway of s-triazine herbicides, were measured for Pseudomonas sp. NRRL B-12227. The rate of degradation was affected by the rate of cyanuric acid transport through cell membranes and the activity of cyanuric acid amidohydrolase inside the cells. At low concentrations of cyanuric acid, the acclimation of cells to cyanuric acid and/or added nutrients effectively enhanced the degradation rate. The strain was also applied to bioremediation using a Bioremediation with Self-Immobilization System (BSIS), in which Pseudomonas sp. NRRL B-12227 cells were co-immobilized with Bacillus subtilis, the latter of which secretes a viscous polymer, in a shallow layer of soil packed in a column. More than 70% of the Pseudomonas sp. NRRL B-12227 cells were co-immobilized with the B. subtilis in a 7.5 cm layer of the packed soil by self-aggregation. More than 60% of the 1 mM cyanuric acid supplied to the packed soil was degraded in this layer during a 72 h period.

Improvement of growth stability of photosynthetic bacterium Rhodobacter capsulatus.

Using a semicontinuous culture method, in which operational parameters such as cell concentration and light intensity distribution were maintained almost constant, instability of the specific growth rate of Rhodobacter capsulatus B-100, a purple bacterium, was observed to be similar to that of R. capsulatus ST-410 when cultivated under high ratios of light intensity on the illuminated side to that of the transmitted light. Such instability was not observed in the cultivation of Chlorella vulgaris, a eukaryotic green alga, even at higher cell concentrations. Under the same conditions, the increase in only the ferrous concentration from 43 microM, the concentration in the original RCV medium, to 172 microM sustained a stable growth, whereas Fe(2+) was slightly consumed during the cultivation. Supplemental illumination with a fluorescent lamp on the transmitted side of a flat plate photobioreactor sustained a moderate level of stable growth, while a halogen lamp slightly affected the growth stability. Our results showed that an increase in Fe(2+) concentration or supplemental illumination improves the growth stability of R. capsulatus.

Fed-batch culture under illumination with blue light emitting diodes (LEDs) for astaxanthin production by Haematococcus pluvialis.

To increase the cell concentration and the accumulation of astaxanthin, the effects of the fed-batch addition of 10-fold-concentrated medium to supply nutrients, as well as illumination with blue light emitting diodes (LEDs), on cell growth and accumulation of astaxanthin were studied for the cultivation of Haematococcus pluvialis. Using the fed-batch addition method, the cell concentration increased above 1 mg-dry cell/cm3, and under illumination with blue LEDs, the astaxanthin concentration reached approximately 70 microg/cm3. This method was much simpler to operate than the medium replacement method in operation and enabled us to attain a higher total yield of astaxanthin.

Effects of temperature on the astaxanthin productivity and light harvesting characteristics of the green alga Haematococcus pluvialis.

The green alga Haematococcus pluvialis, which accumulates astaxanthin at an optimal temperature of 20°C, was cultivated under temperatures of 20°C, 23.5°C, 27°C, and 30.5°C, in order to assess the effects on algal metabolism during the growth phase. The culture growth rate declined with above-optimal increases in temperature, and the final maximum cell concentration at 30.5°C reached only 35% of that attained at 20°C. On the contrary, the biomass productivity was increased under all the high-temperature conditions, probably reflecting the metabolism switch from cell duplication to energy accumulation that is typically observed in algal cultures subjected to environmental stress. Moreover, an increase in the light-harvesting capability of the alga was observed by means of the total pigment balance and the photosynthesis-intensity (PI) curve measured under the different cultivation conditions. Cultures kept at higher temperatures were able to better harvest and utilize the impinging light due to photo-acclimation. Finally, the differences in the astaxanthin metabolism were elucidated by subjecting the cultures to nitrogen starvation at 20°C and 27°C. In the culture at 27°C, a 1.4-fold increase in the astaxanthin productivity was observed when compared to that at 20°C, and the latter required almost two-fold more energy for the astaxanthin production compared with the 27°C culture.