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.

Improving the Stability of Protein-Protein Interaction Assay FlimPIA Using a Thermostabilized Firefly Luciferase.

The protein-protein interaction assay is a key technology in various fields, being applicable in drug screening as well as in diagnosis and inspection, wherein the stability of assays is important. In a previous study, we developed a unique protein-protein interaction assay "FlimPIA" based on the functional complementation of mutant firefly luciferases (Fluc). The catalytic step of Fluc was divided into two half steps: D-luciferin was adenylated in the first step, while adenylated luciferin was oxidized in the second step. We constructed two mutants of Fluc from Photinus pyralis (Ppy); one mutant named Donor is defective in the second half reaction, while the other mutant named Acceptor exhibited low activity in the first half reaction. To date, Ppy has been used in the system; however, its thermostability is low. In this study, to improve the stability of the system, we applied Fluc from thermostabilized Luciola lateralis to FlimPIA. We screened suitable mutants as probes for FlimPIA and obtained Acceptor and Donor candidates. We detected the interaction of FKBP12-FRB with FlimPIA using these candidates. Furthermore, after the incubation of the probes at 37°C for 1 h, the luminescence signal of the new system was 2.4-fold higher than that of the previous system, showing significant improvement in the stability of the assay.

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.

Evaluation of protein-ligand interactions using the luminescent interaction assay FlimPIA with streptavidin-biotin linkage.

Post-translational modifications, such as phosphorylation, are crucial in the regulation of protein-protein interactions and protein function in cell signaling. Here, we studied the interaction between the transactivation domain peptide of cancer suppressor protein p53 and its negative regulator Mdm2 using a novel protein-protein interaction assay, based on the modified FlimPIA using the streptavidin-biotin interaction to link the p53 peptide and the probe enzyme. We succeeded in detecting an attenuation in the affinity of p53 towards Mdm2 caused by the phosphorylation at Thr18. It showed that the targets, which are not easy to fuse with the FlimPIA probes, such as phosphorylated peptides can be used in this system. Also, the use of streptavidin nanobeads was found effective to get clearer signal, probably due to concentration of the detection system onto the bead surface. The system was further applied to the detection of FKBP-FRB interaction using biotinylated FKBP domain, which suggested another potential merit of this system that allows to avoid misfolding and steric hindrance often observed for the fusion protein approach.

Modification of N-Terminal Amino Acids of Fungal Benzoate Hydroxylase (CYP53A15) for the Production of p-Hydroxybenzoate and Optimization of Bioproduction Conditions in Escherichia coli.

The aromatic compound p-hydroxybenzoate (PHBA) is an important material with multiple applications, including as a building block of liquid crystal polymers in chemical industries. The cytochrome P450 (CYP) enzymes are beneficial monooxygenases for the synthesis of chemicals, and CYP53A15 from fungus Cochliobolus lunatus is capable of executing the hydroxylation from benzoate to PHBA. Here, we constructed a system for the bioconversion of benzoate to PHBA in Escherichia coli cells coexpressing CYP53A15 and human NADPH-P450 oxidoreductase (CPR) genes as a redox partner. For suitable coexpression of CYP53A15 and CPR, we originally constructed five plasmids in which we replaced the N-terminal transmembrane region of CYP53A15 with a portion of the N-terminus of various mammalian P450s. PHBA productivity was the greatest when CYP53A15 expression was induced at 20°C in 2×YT medium in host E. coli strain ΔgcvR transformed with an N-terminal transmembrane region of rabbit CYP2C3. By optimizing each reaction condition (reaction temperature, substrate concentration, reaction time, and E. coli cell concentration), we achieved 90% whole-cell conversion of benzoate. Our data demonstrate that the described novel E. coli bioconversion system is a more efficient tool for PHBA production from benzoate than the previously described yeast system.

Improved sensitivity of firefly luminescent intermediate-based protein interaction assay using Ser 440 mutant with lower adenylation activity.

Protein-protein interaction assays are important in various fields including molecular biology, diagnostics, and drug screening. We recently designed a novel protein-protein interaction assay, the firefly luminescent intermediate-based protein interaction assay (FlimPIA), that exploited the unique reaction mechanism of firefly luciferase (Fluc). Using two mutant Flucs, each impaired with one of the two half-reactions, namely adenylation and subsequent oxidative luminescent steps, FlimPIA detects the proximity of the two proteins tethered to the mutant Flucs. Here, we found that introducing a mutation into a residue in the hinge region (S440) of the mutant with lowered adenylation activity ('Acceptor' Fluc) further improved the response of FlimPIA by lowering the residual adenylation activity. Mutants with bulkier residues showed greater inhibition, probably due to increased steric hindrance at the adenylation conformation. As a result, the FlimPIA with S440 L acceptor showed the best signal/background ratio for the detection of rapamycin-induced FKBP12-FRB interactions.

Modifications of a signal sequence for antibody secretion from insect cells.

Monoclonal antibodies and antibody fragments have recently been developed for use in diverse diagnostic and therapeutic applications. Insect cells can efficiently secrete recombinant proteins such as antibody molecules through post-translational processing and modifications that are similar to those performed in mammalian cells. In eukaryotic cells, the signal sequence in a nascent polypeptide is recognized by the signal recognition particle, and the polypeptide is then folded and modified in the endoplasmic reticulum. The signal sequence consists of three regions, a positively charged N-terminus, a hydrophobic core, and a polar C-terminus. In the present study, we examined the substitutions of the characteristic amino acids of a Drosophila immunoglobulin heavy chain binding protein signal sequence, and investigated the effect on the secretory production of an antibody Fab fragment from lepidopteran insect cells in transient expression. A modification of the signal sequence for the heavy chain resulted in a twofold increase in the secreted Fab fragment, while the modification for the light chain led to a more than 3.6-fold increase.

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.

Electrostatic engineering of the interface between heavy and light chains promotes antibody Fab fragment production.

Monoclonal antibodies and antibody fragments are used for diverse diagnostic and therapeutic applications. We have investigated the secretory production of Fab fragments from insect cells cotransfected with plasmid vectors carrying heavy- and light-chain genes. In the present study, to promote the formation of the disulfide bond between the heavy and light chains, some positively charged amino acid residues were introduced near the cysteine residue for the disulfide bond at the C-terminus of CL, while some negatively charged amino acid residues were added near the cysteine residue for the disulfide bond at the C-terminus of CH1. This electrostatic steering led to an increase in Fab secretions from insect cells.

Production of Japanese Encephalitis Virus-Like Particles Using Insect Cell Expression Systems.

Virus-like particles (VLPs) can be produced via the expression of virus surface proteins that self-assemble into particulate structures in recombinant protein expression systems. Expression of the DNA fragment encoding the Japanese encephalitis (JE) virus prM signal peptide, the precursor (prM) of the viral membrane protein (M), and the envelope glycoprotein (E) allows the production of a secretory form of VLPs. Expression systems that use lepidopteran insect cells, such as the baculovirus-insect cell system and stably transformed insect cells, can be used for the efficient production of JE VLPs. This chapter describes the production of JE VLPs from stably transformed lepidopteran insect cells.

Ultra sensitive firefly luciferase-based protein-protein interaction assay (FlimPIA) attained by hinge region engineering and optimized reaction conditions.

Detecting and assaying protein-protein interactions are significant research procedures in biology and biotechnology. We recently reported a novel assay to detect protein-protein interaction, i.e. firefly luminescent intermediate-based protein-protein interaction assay (FlimPIA) using two mutant firefly luciferases (Flucs), which complement each other's deficient half reaction. This assay detects neighboring of two mutant Flucs, namely, a "Donor" that catalyzes the adenylation of firefly luciferin to produce a luciferyl-adenylate intermediate, and an "Acceptor" that catalyzes the subsequent light emitting reaction. However, its rather high background signal, derived from the remaining adenylation activity of the Acceptor, has limited its usefulness. To reduce this background signal, we introduced a mutation (R437K) into the hinge region of the Acceptor, while maintaining the oxidative activity. Interestingly, the signal/background (S/B) ratio of the assay was markedly improved by the addition of coenzyme A and reduction of the ATP concentration, probably due to reduced inhibition by dehydroluciferyl-adenylate formed during the catalysis and an increased ATP-based Km value of the Acceptor, respectively. As a result, a significantly improved maximal S/B ratio from 2.5 to ∼40 was attained, which promises wider use of the assay in in vitro diagnostics, drug discovery, and expanding our knowledge of various biological phenomena.

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.

Suitability and perspectives on using recombinant insect cells for the production of virus-like particles.

Virus-like particles (VLPs) can be produced in recombinant protein production systems by expressing viral surface proteins that spontaneously assemble into particulate structures similar to authentic viral or subviral particles. VLPs serve as excellent platforms for the development of safe and effective vaccines and diagnostic antigens. Among various recombinant protein production systems, the baculovirus-insect cell system has been used extensively for the production of a wide variety of VLPs. This system is already employed for the manufacture of a licensed human papillomavirus-like particle vaccine. However, the baculovirus-insect cell system has several inherent limitations including contamination of VLPs with progeny baculovirus particles. Stably transformed insect cells have emerged as attractive alternatives to the baculovirus-insect cell system. Different types of VLPs, with or without an envelope and composed of either single or multiple structural proteins, have been produced in stably transformed insect cells. VLPs produced by stably transformed insect cells have successfully elicited immune responses in vivo. In some cases, the yield of VLPs attained with recombinant insect cells was comparable to, or higher than, that obtained by baculovirus-infected insect cells. Recombinant insect cells offer a promising approach to the development and production of VLPs.

Production of Japanese encephalitis virus-like particles in insect cells.

Virus-like particles (VLPs) are composed of one or several recombinant viral surface proteins that spontaneously assemble into particulate structures without the incorporation of virus DNA or RNA. The baculovirus-insect cell system has been used extensively for the production of recombinant virus proteins including VLPs. While the baculovirus-insect cell system directs the transient expression of recombinant proteins in a batch culture, stably transformed insect cells allow constitutive production. In our recent study, a secretory form of Japanese encephalitis (JE) VLPs was successfully produced by Trichoplusia ni BTI-TN-5B1-4 (High Five) cells engineered to coexpress the JE virus (JEV) premembrane (prM) and envelope (E) proteins. A higher yield of E protein was attained with recombinant High Five cells than with the baculovirus-insect cell system. This study demonstrated that recombinant insect cells offer a promising approach to the high-level production of VLPs for use as 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.

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.