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.

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.

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.

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 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.

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.

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.

Baculovirus display of functional antibody Fab fragments.

The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

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.

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.

Production of antibody fragments in Escherichia coli.

Escherichia coli is a host widely used in the industrial production of recombinant proteins. However, the expression of heterologous proteins in E. coli often encounters the formation of inclusion bodies, which are insoluble and nonfunctional protein aggregates. For the successful production of antibody fragments, which includes single-chain variable fragments (scFvs), we describe here the modification of linker, signal, and Shine-Dalgarno (SD) sequences, the coexpression of cytoplasmic and periplasmic chaperones, and a method for fed-batch cultivation with exponential feed.

Production of Japanese encephalitis virus-like particles using the baculovirus-insect cell system.

The production of a secreted form of Japanese encephalitis (JE) virus-like particles (VLPs) using the baculovirus-insect cell system was investigated. A recombinant baculovirus that contained the JE virus (JEV) prM signal sequence and the genes encoding the precursor (prM) of the viral membrane protein (M) and the envelope glycoprotein (E) was constructed. Western blotting and enzyme-linked immunosorbent assay (ELISA) of the culture supernatant showed that Spodoptera frugiperda Sf9 cells infected with the recombinant baculovirus had secreted the E protein. Sucrose density-gradient sedimentation analysis of the culture supernatant suggested that secreted E antigen molecules were in a particulate form. Baculovirus-infected Sf9 cells produced more than a 10-fold higher yield of E antigen than that produced by previously reported recombinant CHO cells. Following infection with a recombinant baculovirus encoding a form of prM with a pr/M cleavage site mutation designed to suppress cell-fusion activity of E, Sf9 cells showed an E antigen yield comparable to a yield obtained with the baculovirus encoding the authentic form of prM. Baculovirus-infected Trichoplusia ni BTI-TN-5B1-4 (High Five) cells secreted less of the E antigen than Sf9 cells. Moreover, the Drosophila BiP signal sequence gave an E antigen yield comparable to the prM signal sequence, while the honeybee melittin signal sequence and the baculovirus gp64 signal sequence resulted in lower yields of the E antigen. These results provide information important to the development of VLP production processes using the baculovirus-insect cell system.

Effects of cytoplasmic and periplasmic chaperones on secretory production of single-chain Fv antibody in Escherichia coli.

The effects of cytoplasmic and periplasmic chaperones on the secretory production of an anti-bovine ribonuclease A single-chain variable fragment (scFv) 3A21 in Escherichia coli were investigated. Co-expression of a cytoplasmic chaperone, GroEL/ES, DnaK/DnaJ/GrpE, trigger factor, or SecB with 3A21 scFv affected the proportions of antigen-binding activity in the cytoplasmic soluble fraction, the periplasmic fraction, and the extracellular medium, but there was no significant difference in the total activity compared to the control without chaperone co-expression. On the other hand, co-expression of a periplasmic chaperone, Skp or FkpA, with the exception of DsbC, greatly increased the binding activity in all the soluble fractions. Co-expression of both Skp and FkpA had no synergistic effect. Combinations of cytoplasmic and periplasmic chaperones decreased the productivity. In shake-flask cultures of cells co-expressing Skp or FkpA, considerable amounts of 3A21 scFv were detected in the extracellular medium by enzyme-linked immunosorbent assay (ELISA) and Western blot, and the extracellular production level of 3A21 scFv was calculated to be around 40mg/l. The binding activity of 3A21 scFv co-expressed with Skp was slightly higher than that with FkpA. These results indicate that the co-expression of periplasmic chaperones Skp and FkpA is extremely useful for the secretory production of scFvs in a culture medium using E. coli, but cytoplasmic chaperones and multiple-chaperone combinations may not be effective.

Efficient production of an antibody Fab fragment using the baculovirus-insect cell system.

The production of an Fab fragment of the catalytic antibody 6D9 in lepidopteran insect cells infected with a recombinant baculovirus that contained both the heavy chain (Hc) and light chain (Lc) genes of the Fab fragment was investigated. Western blotting and enzyme-linked immunosorbent assay (ELISA) of culture supernatant showed that baculovirus-infected Trichoplusia ni BTI-TN-5B1-4 (High Five) cells secreted an Fab fragment that retained antigen-binding activity. Infection of High Five cells with a recombinant baculovirus, in which the Lc and Hc genes were located downstream of the baculovirus p10 and polyhedrin promoters, respectively, produced a higher Fab fragment yield than that obtained with a baculovirus in which the Hc and Lc genes were downstream of the p10 and polyhedrin promoters, respectively. Baculovirus-infected High Five cells secreted more of the Fab fragment than Spodoptera frugiperda Sf9 cells. Moreover, use of the baculovirus gp64 signal sequence upstream of the Lc and Hc genes resulted in greater yield of the secreted Fab fragment than use of the insect-derived BiP and melittin signal sequences. Consequently, the Fab fragment was obtained in a high yield (>600 µg/ml) in a shake-flask culture of High Five cells infected at a multiplicity of infection (MOI) of 10 plaque-forming units (pfu)/cell with the recombinant baculovirus in which the Lc and Hc genes with the gp64 signal sequence were expressed under the control of the p10 and polyhedrin promoters, respectively. These results indicate that the baculovirus-insect cell system may allow efficient production of antibody Fab fragments.

Immobilization of 293 cells using porous support particles for adenovirus vector production.

Adenovirus vector production by anchorage-independent 293 cells immobilized using porous biomass support particles (BSPs) was investigated in static and shake-flask cultures for efficient large-scale production of adenovirus vectors for gene therapy applications. The density of cells immobilized within BSPs was evaluated by measuring their WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) reduction activity. In shake-flask culture, 293-F cells, which were adapted to serum-free suspension culture, were not successfully retained within reticulated polyvinyl formal (PVF) resin BSPs (2 × 2 × 2 mm cubes) with matrices of relatively small pores (pore diameter 60 µm). When the BSPs were coated with a cationic polymer polyethyleneimine, a high cell density of more than 10(7) cells cm(-3)-BSP was achieved in both static and shake-flask cultures with regular replacement of the culture medium. After infection with an adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP), the specific Ad EGFP productivity of the immobilized cells was comparable to the maximal productivity of non-immobilized 293-F cells by maintaining favorable conditions in the culture environment.

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.

Adenovirus vector production using low-multiplicity infection of 293 cells.

The use of low-multiplicity infection of 293 cells in static culture with regular medium replacement was investigated for efficient large-scale production of adenovirus vectors for gene therapy applications. An adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP) was used to infect 293-F cells at a low multiplicity of infection (MOI) of 0.00001-0.1 transductional unit (TU) cell(-1). The cells, which have the ability to grow in suspension, were incubated in T-flasks and the serum-free culture medium was replaced with fresh medium via centrifugation every 2 days. Because only a small proportion of cells were initially infected at low MOIs (<1 TU cell(-1)), uninfected cells continued to grow until they were infected by progeny adenoviruses released from previously infected cells. When 293-F cells at a relatively low density of 1 x 10(5) cells cm(-3) were infected with Ad EGFP at a low MOI of 0.001 TU cell(-1), the vector yield was 2.7-fold higher than the maximum yield obtained with high-multiplicity infection (MOI = 10 TU cell(-1)) in batch culture. These results indicate that efficient adenovirus vector production using low MOIs is achieved by minimization of either nutrient depletion and/or accumulation of inhibitory metabolites in the culture medium.

Tandem multimer expression of angiotensin I-converting enzyme inhibitory peptide in Escherichia coli.

It is common for small tandem peptide multimer genes to be indirectly inserted into expression vectors and fused with a protein tag. In this study, a multimer of the tandem angiotensin I-converting enzyme inhibitory peptide (ACE-IP) gene was directly transferred to a commercially available vector and the designed gene was expressed as a repeated peptide in Escherichia coli BL21(DE3)pLysS. The process further developed in our study was the construction of six-repeated ACE-IP synthetic genes and their direct insertion. Protein expression in inclusion bodies was confirmed by SDS-PAGE and Western blot. Acid hydrolysis of inclusion bodies produced single-unit peptides through cleavage of the aspartyl-prolyl bonds. This cleaved recombinant peptide (rACE-IP) was purified using immuno-affinity chromatography followed by reversed phase-HPLC. 105-115 mg of the lyophilized recombinant peptide was obtained from 1 L E. coli culture. In vitro biological activity of rACE-IP was indistinguishable from that of the natural peptide produced by hydrolysis in artificial gastric juice or by acidic hydrolysis. The rACE-IP prepared by recombinant DNA technology and solid-phase synthesis methods showed a similar IC(50). This strategy could be used for the expression of important peptides, which have N-terminal proline (P) and C-terminal aspartic acid residues (D) for commercial applications, e.g. functional foods and drinks.

Angiotensin-I converting enzyme (ACE) inhibitory mechanism of tripeptides containing aromatic residues.

Angiotensin-I converting enzyme (ACE) plays important roles in the regulation of blood pressure, and ACE inhibitory peptides in food materials have attracted attention for their antihypertensive function. In this study, the function of amino acids in ACE inhibitory tripeptides was clarified.

High efficiency production of astaxanthin in an airlift photobioreactor.

In photobioreactors, photosynthetic microorganisms are exposed to certain light/dark cycles caused by light intensity distribution and mixing inside the photobioreactor. In this study, Haematococcus pluvialis was cultivated in an airlift and a bubble column photobioreactor, and the cell growth and astaxanthin production were compared to clarify the effects of liquid circulation.