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Olfactory receptor (OR) genes are considered to be the largest superfamily of the mammalian genome, and in the case of humans, approximately 390 kinds of functional ORs play a role in perceiving odors. In spite of their significance in olfaction, the function of all ORs has not yet been fully revealed. In order to efficiently identify specific ligands of orphan ORs, methods that can generate olfactory signals in a reliable manner and that can convert the cellular signals into measurable responses are required. Here, we describe an OR screening assay method using olfactory sensors that are based on cell-derived nanovesicles combined with single-walled carbon nanotube field-effect transistors (SWNT-FETs). The nanovesicles contain ORs on their surface membrane and induce influx of calcium ions similar to olfactory signal transduction. This ion influx causes an electrical current change along the carbon nanotube, and then this change is measured by the SWNT-FET sensor. This technique facilitates the simple and rapid screening of OR functions.

Silkworm hemolymph (SH) has been reported to inhibit apoptosis in both insect and human cells, and increase the high-sialylation structure of recombinant glycoprotein in insect cells. This indicates that SH might increase glycosyltransferase activity. Therefore, this study examined the effect of SH on the activity of sialyltransferase, which catalyzes the sialylation of the glycoprotein. When 10 μg/mL of SH was added to the reaction mixture, almost complete sialylation was observed even under the reaction conditions where sialyltransferase-catalyzed sialylation rarely occurs. The effect of deproteinized SH (dSH) and the 30K protein, which is a major plasma protein in SH, was examined to determine which component in SH enhances sialylation. The 30K protein promoted sialylation, while the dSH did not. This suggests that SH and its 30K protein can be used as an additive to a medium for efficient glycosylation when mammalian cells are being cultured for the production of valuable biopharmaceuticals, many of which are glycoproteins.

An alternative microalgal system for biological wastewater treatment is proposed for both the removal of nitrogen and phosphorus from wastewater and the production of a valuable carotenoid, astaxanthin. The system consists of sequential photoautotrophic cultivation and induction processes using the green alga Haematococcus pluvialis . The Haematococcus process was applied to primary-treated sewage (PTS) and primary-treated piggery wastewater (PTP) with serial dilution. H. pluvialis grew well on PTS and PTP diluted four-fold, resulting in the successful removal of nitrogen and phosphorus from both wastewaters. At that time, cell growth rates were comparable to those in the algal-defined NIES-C medium. Following the cultivation stage, N-deprived vegetative cells were transformed under photoautotrophic induction by continuous feeding of both CO 2 -mixed gas and intense light to red aplanospores with substantial astaxanthin contents. The resulting astaxanthin contents accounted for about 5.1 and 5.9% of the total biomass of the PTS and PTP cultures, respectively. Our results indicate the potential of the proposed Haematococcus process as a subsidiary wastewater treatment technology with the capability of biosynthesizing the high-value antioxidant astaxanthin.

Chlamydomonas reinhardtii is a green alga that can use light energy to produce hydrogen from water under anaerobic conditions. This work reports the enhancement of hydrogen production by controlling the light intensity in sulfur-deprived anaerobic C. reinhardtii cultures. The overall hydrogen production was dependent on light intensity in the range of 60-200 μE m -2 s -1 . Maximum hydrogen production was obtained at a light intensity of 200 μE m -2 s -1 as a result of the rapid initiation of hydrogen production and the greatest increase of chlorophyll during the initial 24 h after sulfur deprivation. However, the hydrogen production was inhibited at an intensity of 300 mE m -2 s -1 of light owing to photosystem II photodamage by excess light. The maximum hydrogen production and the maximum specific production rate of hydrogen were 225 ml H 2 l -1 culture and 2.01 ml H 2 g -1 cells h -1 , respectively. Thus, hydrogen production by sulfur-deprived C. reinhardtii cultures can be maximized by controlling the light intensity at levels below saturation.

The disadvantage of a bacteriophage λ expression system is that the replicated λ DNA containing a foreign gene is coated by a phage head, and cell lysis occurs before sufficient expression of the cloned gene. The late genes of the bacteriophage λ system are related to λ DNA packaging and host cell lysis. This study investigated the late gene mutants of the bacteriophage λ in an attempt to develop an efficient expression vector system. The bacteriophage λNM1070, which is a W − , E − and S − mutant, had increased cell longevity due to the S − mutation. However, the genetic stability of this expression system was not high enough because the W − and E − mutation caused a defect in its re-infection. The bacteriophage λHL1, which is a Q − mutant, represses the transcription of all the late genes, even though this repression was not complete. This incomplete repression was rather beneficial to the cloned gene expression because it produced some matured phage particles, which re-infected the segregated host cell and increased the genetic stability. Therefore, a double mutant of the genes S and Q was constructed in order to exploit the merits of λNM1070 and λHL1. This λ Q − S − mutant had higher host cell longevity and cloned gene expression than the λNM1070 and λHL1.

Surface plasmon resonance (SPR) biosensor based on mixed self-assembled monolayers (SAMs) was developed for real-time detection of Cryptosporidium oocyst. The flow type SPR biosensor consisting of subsequent binding of antibody and oocyst in flowing buffer system was able to detect Cryptosporidium parvum oocyst in real-time with a detection limit of 1 × 106 oocyst/ml. To enhance the sensitivity of real-time detection, the modified flow type SPR biosensor was developed, which consisted of the immunoreaction step between the biotin-antibody and oocysts followed by the binding step of antibody bound oocysts onto the sensor surface. Not only was the immunoreaction yield with batch immune reaction improved, but the accessibility of analytes to the sensor surface was also enhanced since the binding event for SPR signal generation was exchanged to biotin–streptavidin binding with high affinity. Consequently, the real-time detection limit of 1 × 102 oocyst/ml was obtained in the modified flow type biosensor.

The surface plasmon resonance (SPR) technique has been used for characterizing molecular interactions. Recently, this technique was used for the analysis of interactions between living cells and molecules reactive to cells. In this study, the technique was applied to the cell-based measurement of odorant molecules. HEK-293 cells were used as a heterologous cell system, and ODR-10, the olfactory receptor of the nematode Caenorhabditis elegans, as a model olfactory receptor. The ODR-10 was expressed on the cell surface, with the help of the rho-tag import sequence, which was fused at the N-terminus of the ODR-10. Exposure of the cells to 0.1 mM diacetyl, which is an odorant molecule specific to the ODR-10, induced a SPR signal from the HEK-293 cells expressing ODR-10, while no SPR signal was detected from the control HEK-293 cells. The intensity of the induced signal was dependent on the dose of diacetyl. These SPR signals were regarded as a result of the intracellular signaling triggered by the binding of odorant molecules to the olfactory receptors. This method, using a heterologous olfactory cell system and SPR system, can be efficiently used to identify the odorant molecules specific to each olfactory receptor.

Cyanobacteria provide an efficient system for producing H 2 from water using solar energy. The energy conversion efficiency can be defined by the ratio of H 2 produced to the light energy absorbed. An IR and opalescent plate method was used to measure the light energy absorbed. Since cyanobacteria absorb light in the visible range but not in the infrared range, the net amount of light energy absorbed by the cells can be estimated by measuring the IR and visible light intensities transmitted through the biochamber. A rectangular biochamber was used for measuring the conversion efficiency from light energy to H 2 energy. A quantum meter and radiometer were used to measure the light intensity transmitted through the chamber. Anabaena variabilis was cultured in a BG11 medium with 3.6 mM NaNO 3 and the light intensity was 40–50 μ mol / m 2 / s in the growth phase and 120–140 μ mol / m 2 / s in the H 2 production phase. The maximum H 2 production was 50 ml for 40 h and cell density was 1.2 g/l. The H 2 production rate was 4.1 ml H 2 / g dry cell weight/h. Based on the light absorbed in the H 2 production phase, the energy conversion efficiency from light to H 2 was 1.5% on average and 3.9% at the maximum. Based on the light energy absorbed in the cell growth and H 2 production phases, the energy conversion efficiency was 1.1% on average.

Polymerase chain reaction (PCR) is an important molecular biological tool for the amplification of nucleic acids. PCR process can be divided into three phases according to the amplification rate: exponential, quasi-linear, and plateau. We investigated the cause of the plateau phenomenon through real-time monitoring of the amplification profile and computerized simulation. Possible limiting components, such as Taq DNA polymerase, primer pair, and dNTPs were added during quasi-linear phase, after which the differences in the amplification profiles were monitored. Modeling and computerized simulations were performed to look into the complex mechanism of the reactions, such as renaturation of templates during temperature transition from denaturation to annealing step and effective enzyme concentration profiles within the cycle progress. The decrease of effective polymerase concentration due to heat inactivation and product accumulation caused the PCR plateau. Addition of polymerase during the quasi-linear phase could increase the final product amount; however, the PCR process still reached the plateau phase in spite of polymerase addition. Simulation results suggest that renaturation of templates before competitive annealing reaction and decrease of effective enzyme concentration by non-specific binding of polymerase to double-stranded DNA is the main contribution to plateau forming.

A continuous-flow (CF) microchip was fabricated using polydimethylsiloxane (PDMS)/glass laminate for efficient polymerase chain reaction (PCR). The temperatures of the three separate zones, corresponding to the three PCR thermal cycling steps were controlled using an external PID temperature controller. A PCR mixture was introduced to the CF microchip using a syringe pump. The hydrophobic surface of the PDMS channel which adsorbs enzymes was modified by adding 2.5% polyvinylpyrrolidone (PVP) to the PCR mixture. The effect of flow rate in the CF chip on DNA amplification was examined and its PCR efficiency was compared with that of conventional PCR. The microchip requires only 8–30 min to produce detectable amounts of PCR products as determined by slab gel separation, whereas a conventional thermocycler requires 90–150 min over 30 amplification cycles to achieve the same result.

It was previously reported that silkworm hemolymph (SH) inhibits apoptosis and increases the production of recombinant human erythropoietin (EPO) in Chinese hamster ovary (CHO) cells. The apoptosis-inhibiting component in SH is a member of 30K protein family. In this study, the CHO cell line producing EPO was manipulated genetically to express the 30Kc6 gene encoding a 30K protein in the hemolymph of the silkworm, Bombyx mori. The transient expression of 30Kc6 significantly suppressed the cell death induced by serum deprivation. A stable cell line expressing 30Kc6 with an anti-apoptotic property was established. The stable expression of 30Kc6 inhibited serum-deprivation-induced apoptosis and increased the cell density and EPO titer by 5- and 10-fold, respectively. The positive effects of the 30Kc6 expression on cell viability and productivity were due to the stable maintenance of the mitochondrial activity. The 30Kc6 expression efficiently suppressed the depolarization of the mitochondrial membrane and subsequently balanced the generation/consumption of ATP. The use of the 30Kc6 gene is expected to provide a new method of host cell engineering for improving the productivity of the recombinant protein.

Controversy exists over the relationship between the cAMP and IP3 pathways in vertebrate olfactory signal transduction, as this process is known to occur by either of the two pathways. Recent studies have shown that a single olfactory neuron responds to both cAMP- and IP3-producing odorants, suggesting the existence of an olfactory receptor protein that can recognize both ligands. In this study we found that the rat olfactory receptor I7, stably expressed in HEK-293 cells, triggers the cAMP pathway upon stimulation by a specific odorant (octanal) at concentrations lower than 10-4 M; however, the receptor triggers both pathways at higher concentrations. This indicates that a single olfactory receptor, stimulated by a single pathway-inducing odorant, can evoke both pathways at high odorant concentrations. Using this heterologous system, both the dose-dependent response and receptor I7 specificity were analyzed. The dose-dependent Ca2+ response curve, which also includes the release of Ca2+ ions from internal stores at high odorant concentrations, was not monotonous, but had a local maximum and minimum with 10-10 and 10-7 M octanal, respectively, and reached a plateau at 10-2 M octanal. The specificity of the I7 receptor was lower when exposed to higher concentrations of odorants.

A two-stage continuous culture of Escherichia coli in combination with a bacteriophage λ system was performed in order to overcome the intrinsic plasmid instability that is frequently observed in recombinant fermentation. A phage λ vector with a Q − mutation was used to enhance the expression of the λ system. The optimal values of the important operational variables such as the substrate concentration, the dilution rate, and the mean residence time on the expression of the cloned gene were determined in both batch and continuous cultures. For all culturing modes, the full induction of the cloned gene was observed 4 h after the temperature shift. In the two stage continuous culture, the overproduction reached their maxima at D=0.25 h−1 with 1.5 S 0 of the medium supply. The maximum productivity of the total β-galactosidase was 16.3×106 U l−1 h−1, which was approximately seven times higher than that in the single-copy lysogenic stage. The recombinant cells were stable in the lysogenic state for more than 260 h, while they were stable for 40 h in the lytic state. The instability that developed rapidly in the second tank is believed to be due to the accumulation of lysis proteins as a result of vector leakage during the operation.

The effect of silkworm hemolymph on the expression of recombinant protein inEscherichia coli was investigated. The addition of silkworm hemolymph to the culture medium increased the production of recombinant β-galactosidase inE. coli. The production was dependent on the concentration of the added silkworm hemolymph, which increased 2-, 5-, and 8-fold in media supplemented with 1,3, and 5% silkworm hemolymph, respectively. To identify the effective component, the silkworm hemolymph was fractionated by gel filtration column chromatography. A fraction, with a molecular weight of about 30 K was identified as the effective component.

The first AFOB special issue of Biotechnology Journal is edited by Prof. Tai Hyun Park and Prof. George G. Q. Chen. The eleven articles are representative of the diverse nature of biotechnology today, covering topics such as microfluidic devices, high-throughput analysis, biosensors, bio-imaging, tissue engineering, vaccination, gene delivery, gene expression, and cell-free protein synthesis.

Enzymatic in vitro glycosylation is possible using a reverse reaction of peptide-N-glycosidase F (PNGase F), and non-enzymatic in vitro glycosylation occurs when the sugar residue is one or two units long. To identify the differences between enzymatic and non-enzymatic glycosylation, glycosylation sites were analyzed by the acid hydrolysis of glycopeptides followed by MALDI-TOF mass spectrometric analysis. Pentapeptide (Arg-Lys-Asp-Val-Tyr) and octapeptide (Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr) were used in this study, and the sequence of the octapeptide was appropriately chosen to investigate the specificity of enzymatic glycosylation by considering the characteristics of PNGase F and non-enzymatic glycosylation. N,N′-Diacetylchitobiose was aminated prior to the glycosylation reaction at an amination extent of 60%. The glycosylation site was very specific to the aspartate residue in the enzymatic reaction, while non-enzymatic glycosylation occurred at arginine or lysine residues. PNGases F can be effectively used for the glycosylation of the non-glycosylated recombinant proteins produced in prokaryotic cells.

A recombinant baculovirus was constructed by the homologous recombination between wild-type AcMNPV DNA and a baculovirus transfer vector containing a gene coding for the 30K protein originating from silkworm hemolymph. The 30K protein was successfully expressed in Sf9 cells infected with the recombinant baculovirus (AcMNPV/30K). To investigate the effect produced by the expression of the 30K protein, host cell viability after infection was compared with that of Sf9 cells infected with AcMNPV/β-gal. The viability of the cells infected with AcMNPV/β-gal began to decrease exponentially 3 days after infection, whereas that of the cells infected with AcMNPV/30K remained at a high level until 5 days after infection. This indicates that the 30K protein increases cell longevity after viral infection. This increased cell longevity is considered to be due to the inhibition of host cell apoptosis induced by a baculovirus, and the extent of apoptosis was measured by the flow cytometric method. The percentage of the sub-G1 fraction, which represents the extent of apoptosis, was decreased by the expression of the 30K protein. This indicates that the expression of the 30K protein in insect cells increases host cell longevity by inhibiting apoptosis.

The cell orientation and attachment were controlled by a submicron-scale topographical cue. For achieving the submicron undulation of surface topography, the laser holography on a photo-responsive azobenzene copolymer layer has been employed to produce surface relief grating (SRG) which has a regular sinusoidal shape. Cultured human astrocytes (HA) were preferentially attached onto the SRG surface and highly elongated along the SRG direction. This topographical control scheme would be very efficient to control the cell growth for cellular engineering applications and to understand the interactions of the cells with a submicron-scale topographical surface.

In a previous study, we reported that silkworm hemolymph inhibits apoptosis and that the anti-apoptotic component in silkworm hemolymph is a 30K protein. In this study, the 30K protein encoded by 30Kc6 was expressed in Escherichia coli . The recombinant 30K protein was expressed as an inclusion body, and the inclusion body was separated and refolded by affinity column chromatography using a 6× His tag. We demonstrated that apoptosis is inhibited by supplementing the culture medium with this purified recombinant 30K protein. The recombinant 30K protein inhibited the virus- or chemical-induced apoptosis in human cells as well as insect cells. Apoptosis-inhibitory activity of recombinant 30K protein was comparable to that of whole silkworm hemolymph. The recombinant 30K protein can be effectively used to minimize cell death and consequently increase the productivity by extending the production time of host cells in commercial animal cell culture.

Polymerase chain reaction (PCR) is one of the most widely used analytical tool and is an important module that would benefit from being miniaturized and integrated onto diagnostic or analytical chips. There are potentially two different approaches for the miniaturization of the PCR module: chamber-type and flow-type micro-PCR. These miniaturized PCRs have distinct characteristics and advantages. In this article, we review the necessity of micro-PCR, the materials for the chip fabrication, the surface modification, and characteristics of the two types of micro-PCR. The motivation underlying the development of micro-PCR, the advantages and disadvantages of the various materials used in fabrication and the surface modification methods will be discussed. And finally, the precise features of the two different types of micro-PCR will be compared.

An increased understanding of apoptosis makes anti-apoptosis engineering possible, which is an approach used to inhibit apoptosis for the purpose of therapeutic, or industrial applications in the treatment of the diseases associated with increased apoptosis, or to improve the productivity of animal cell cultures, respectively. Some known anti-apoptotic proteins are the Bcl-2 family, IAP (inhibitor of apoptosis) and Hsps (heat shock proteins), with which anti-apoptosis engineering has progressed. This article reviews anti-apoptosis engineering using known anti-apoptotic compounds, and introduces a 30 K protein, isolated from silkworm hemolymph, as a novel anti-apoptotic protein, that shows no homology with other known anti-apoptotic proteins. The regulation of apoptosis, using anti-apoptotic proteins and genes originating from the silkworm,Bombyx mori, may provide a new strategy in this field.

Microbial biofouling is one of the major obstacles for reaching the ultimate goal to realize high permeability over a prolonged period of reverse osmosis operation. In this study, the hybrid thin-film-composite (TFC) membrane consisted of self-assembly of TiO2 nanoparticles with photocatalytic destructive capability on microorganisms was devised as a novel means to reduce membrane biofouling. Then, the anti-fouling and fouling mitigation on the actual commercialized TFC was verified. TiO2 nanoparticles of a quantum size (∼10 nm or less) in anatase crystal structure were prepared from the controlled hydrolysis of titanium tetraisopropoxide and characterized by X-ray diffraction (XRD) analysis and transmission electron microscopy (TEM). Hybrid thin-film-composite (TFC) membrane was prepared by self-assembly of the TiO2 nanoparticles through coordination and H-bonding interaction with the COOH functional group of aromatic polyamide thin-film layer, which was ascertained by X-ray photoelectron spectroscopy (XPS). The hybrid membrane was shown to possess the dramatic photobactericidal effect on Escherchia coli (E. coli) under UV light illumination. Finally, introduction of TiO2 nanoparticles on the actual commercial TFC membrane and application of RO field test after exposure to microbial cells verified a substantial prevention against the microbial fouling by showing less loss of RO permeability, offering a strong potential for possible use as a new type of anti-biofouling TFC membrane.

Cyanobacteria have the unique characteristic of using CO 2 in the air as a carbon source and solar energy as an energy source. Reducing equivalents from the fermentation of carbohydrates are used as the primary electron donors in cyanobacteria for the hydrogen producing enzymes. The cells take up CO 2 first to produce cellular substances, which are subsequently used for H 2 production. Since the optimal operating conditions for the CO 2 uptake and H 2 production are different, a two-stage system can be effectively employed to separate these two phases. In this study, for the efficient production of H 2 in the second stage, the conditions for the effective CO 2 uptake and cell growth in the first stage were characterized, and high cell density culture was carried out using repeated injections of CO 2 . The specific growth rate and growth yield based on CO 2 decreased with an increase in light intensity or CO 2 concentration. However, the effect of CO 2 concentration on the growth yield was much smaller than that of a light intensity. A CO 2 uptake rate per unit cell decreased linearly with the initial CO 2 concentration in the gas phase. With repeated injections of CO 2 , the CO 2 was continuously consumed and the cell concentration reached 3.7 g dry cell/l in 20 days, which is 6.7 times higher than that in a batch culture without further supply of CO 2 . The CO 2 injection in the cell growth phase increased not only the cell concentration but also the hydrogen production per gram cell.

We have previously shown that silkworm hemolymph exhibits anti-apoptotic activity against baculovirus-induced Sf9 cell apoptosis. In this study, using various chemical inducers, such as actinomycin D, camptothecin, and staurosporine, we found that silkworm hemolymph inhibits insect cell apoptosis induced not only by baculovirus but also by chemical inducers. This indicates that silkworm hemolymph contains anti-apoptotic components that work directly in insect cell apoptosis without any booster expression of baculoviral genes. With the analysis of Sf-caspase-1 activity, it was found that the inhibitory effect of silkworm hemolymph works in a further upstream step than the Sf-caspase-1 activation step.

A feeding strategy was developed for controlling the specific growth rate in a Bacillus subtilis fed-batch operation, and the importance of the specific growth rate was investigated for subtilisin expression. The ratio of the glucose and peptone concentrations in the feeding medium was critical in controlling the specific growth rate, and the appropriate ratio was found to be 1:2. The specific growth rate was well controlled in fed-batch operations including an exponential feeding of a 200 g/liter glucose and 400 g/liter peptone solution. Controlling the value of the specific growth rate had a significant effect on the subtilisin expression, even though the subtilisin expression was only enhanced after the feeding ended. This would appear to be due to cell components, such as sigma factors, produced in the growth phase that influenced the aprE gene expression. The optimal specific growth rate for the maximum expression rate and longest production period was 0.35 h−1.

A high yield and cost effective process for the production of glycoproteins is attainable based on a low cost prokaryotic system with a high yield production of a nonglycosylated form, which is then glycosylated in vitro. Accordingly, the enzymatic deglycosylation of glycoproteins and enzymatic glycosylation of deglycosylated proteins were investigated using peptide-N-glycosidase F. Enzymatic in vitro glycosylation was found to occur with excess maltodextrin and ammonium salt in acetone at 50°C.

Chondrogenic commitments of mesenchymal stem cells (MSCs) require 3D cellular organization. Furthermore, recent progresses in bioreactor technology have contributed to the development of various biophysical stimulation platforms for efficient cartilage tissue formation. Here, an approach is reported to drive 3D cellular organization and enhance chondrogenic commitment of bone-marrow-derived human mesenchymal stem cells (BM-hMSCs) via magnetic nanoparticle (MNP)-mediated physical stimuli. MNPs isolated from Magnetospirillum sp. AMB-1 are endocytosed by the BM-hMSCs in a highly efficient manner. MNPs-incorporated BM-hMSCs are pelleted and then subjected to static magnetic field and/or magnet-derived shear stress. Magnetic-based stimuli enhance level of sulfated glycosaminoglycan (sGAG) and collagen synthesis, and facilitate the chondrogenic differentiation of BM-hMSCs. In addition, both static magnetic field and magnet-derived shear stress applied for the chondrogenic differentiation of BM-hMSCs do not show increament of hypertrophic differentiation. This MNP-mediated physical stimulation platform demonstrates a promising strategy for efficient cartilage tissue engineering.

The sense of taste helps humans to obtain information and form a picture of the world by recognizing chemicals in their environments. Over the past decade, large advances have been made in understanding the mechanisms of taste detection and mimicking its capability using artificial sensor devices. However, the detection capability of previous artificial taste sensors has been far inferior to that of animal tongues, in terms of its sensitivity and selectivity. Herein, we developed a bioelectronic tongue using heterodimeric human sweet taste receptors for the detection and discrimination of sweeteners with human-like performance, where single-walled carbon nanotube field-effect transistors were functionalized with nanovesicles containing human sweet taste receptors and used to detect the binding of sweeteners to the taste receptors. The receptors are heterodimeric G-protein-coupled receptors (GPCRs) composed of human taste receptor type 1 member 2 (hTAS1R2) and human taste receptor type 1 member 3 (hTAS1R3), which have multiple binding sites and allow a human tongue-like broad selectivity for the detection of sweeteners. This nanovesicle-based bioelectronic tongue can be a powerful tool for the detection of sweeteners as an alternative to labor-intensive and time-consuming cell-based assays and the sensory evaluation panels used in the food and beverage industry. Furthermore, this study also allows the artificial sensor to exam the functional activity of dimeric GPCRs.

In previous studies, 30Kc19, a lipoprotein in silkworm hemolymph, enhanced productivity and glycosylation by expression of a 30Kc19 gene or supplementation with a recombinant 30Kc19 protein. Additionally, 30Kc19 exhibited enzyme-stabilizing and cell-penetrating abilities in vitro. In this study, we hypothesized that supplemented 30Kc19 penetrated into the cell and enhanced the stability of the cellular enzyme. We investigated this using in vitro and cellular assessments. The activity of sialyltransferase (ST) and isolated mitochondrial complex I/III was enhanced with 30Kc19 in dose-dependent manner while initial reaction rate was unchanged, suggesting that 30Kc19 enhanced enzyme stability rather than specific activity. For intracellular enzyme activity assessment, ST activity inside erythropoietin (EPO)-producing Chinese hamster ovary (CHO) cells increased more than 25 % and mitochondrial complex II activity in HeLa cells increased more than 50 % with 30Kc19. The increase in intracellular ST activity resulted in an increase in sialic acid content of glycoproteins produced in CHO cells supplemented with 30Kc19. Similarly, enhanced mitochondrial complex activity increased mitochondrial membrane potential and ATP production in HeLa cells with 30Kc19 by over 50 %. Because 30Kc19 stabilized intracellular enzymes for glycosylation and enhanced protein productivity with supplementation in the culture medium, we expect that 30Kc19 can be a valuable tool for effective industrial recombinant protein production.

Recently, the recombinant 30Kc19 protein, originating from silkworm hemolymph of Bombyx mori has attracted attention due to its cell-penetrating property and potential application as a protein delivery system. However, this observation of penetration across cell membrane has raised questions concerning the interaction of the protein-lipid bilayer. Here, we report a dimerization propensity of the 30Kc19 protein in the presence of amphiphilic moieties; sodium dodecyl sulfate (SDS) or phospholipid. Native PAGE showed that the 30Kc19 monomer formed a dimer when SDS or phospholipid was present. In the glutathione-S-transferase (GST) pull-down assay, supplementation of the 30Kc19 protein to mammalian cell culture medium showed dimerization and penetration; due to phospholipids at the cell membrane, the main components of the lipid bilayer. Mutagenesis was performed, and penetration was observed by 30Kc19 C76A and not 30Kc19 C57A, which meant that the presence of cysteine at position 57 (Cys-57) is involved in dimerization of the 30Kc19 at the cell membrane during penetration. We anticipate application of the native 30Kc19 protein with high cell-penetrating efficiency for delivery of cargos to various cell types. The intracellular cargo delivery using the 30Kc19 protein is a non-virus derived (e.g. TAT) delivery method, which can open up new approaches for the delivery of therapeutics in bioindustries, such as pharma- and cosmeceuticals.

Olfactory receptors, belonging to a family of G protein-coupled receptors (GPCRs), which are involved in various important physiological processes, are integral membrane proteins composed of seven transmembrane helices. It is difficult to produce GPCRs including olfactory receptors (ORs) using heterologous cell systems, because of their strong hydrophobicity, and complicated structure. The production of ORs should be a critical process for the development of an olfactory receptor-based bioelectronic nose. Significant efforts have been made for the production of ORs for the utilization as recognition elements of bioelectronic noses, and also for other applications. In addition, the construction method of mammalian cell-derived nanovesicles containing ORs has been demonstrated and applied for an bioelectronic nose, due to their unique properties, and suitable size for integration with a nanosensor platform. In this chapter, advances in the production of ORs and nanovesicles using various heterologous cell systems for the development of a bioelectronic nose are described.

Detection and discrimination of odorants has great potential for applications in various fields, such as the food industry, fragrance and flavor industry, environmental monitoring, and biomedical diagnosis. For several decades, many efforts have been made to control the process of food production and fragrance and flavor of brands, and to monitor environmental pollutions through the use of comparable technology. There have been several classical methods for these purposes. Conventional methods, such as GC/MS or human sensory panels (olfactometry), have been conventionally used, but they are expensive, labor-intensive, time-consuming and affected by large variations according to the conditions of analysis. These drawbacks increased the requirement for new technique, substituting classical methods, and the electronic nose has been developed over the past couple of decades. However, the electronic nose has also many limitations to be overcome. Recently, the bioelectronic nose, using biological components, has been developed. The bioelectronic nose has a bright prospect as a powerful and effective biosensing system, capable of detecting and discriminating a huge variety of odorant molecules. The most meaningful characteristics of the bioelectronic nose are that it mimics the human olfactory system. The bioelectronic nose is expected to replace the sensory evaluation method. It can be used for standardization of smell, development of code for each smell, and visualization of smell. Consequently, the development of the bioelectronic nose is expected to open up many new possibilities to improve the quality of our life.

Sense of smell is an important sense to recognize environmental conditions and dangerous situations. Following the identification of the olfactory mechanism in the early 1990s, extensive studies to develop electronic devices that mimic the function of animal noses have been conducted. Most devices have been composed of an array of several sensors that react to chemical compounds. The odor is characterized by analyzing the response patterns generated by the sensor array. However, such devices have limitations in terms of sensitivity and selectivity. Hence, a novel concept for sensor devices functionalized with odor-recognizing biomolecules was suggested. Sensors which use biomolecules as a primary sensing material are commonly called bioelectronic noses. A bioelectronic nose generally consists of primary and secondary transducers. The primary transducer is a biological recognition element such as olfactory receptors and odorant-binding proteins. The secondary transducer is a highly sensitive optical or electrical sensor platform that converts biological events into measurable signals. In this chapter, the basic concept and principles of bioelectronic noses are described. In addition, specific characteristics of bioelectronic noses and the current issues are presented.

Silkworm (Bombyx mori) hemolymph showed an apoptosis-inhibiting activity in insect cells (Sf 9) infected with baculovirus (AcNPV). The addition of silkworm hemolymph into the culture medium increased the host cell longevity due to its apoptosis-inhibition activity. Components with an apoptosis-inhibiting effect were purified from the silkworm hemolymph by heat treatment, gel-filtration chromatography, and ion-exchange chromatography. The component with highest activity was characterized by periodic acid–Schiff staining, isoelectric focusing, MALDI-TOF–mass spectrometry, and N-terminal sequencing and was found to be a nonglycosylated monomeric protein with a molecular weight of ca. 28,000 Da.

To develop a novel toxicity measurement system using the persistent swimming property of magnetic bacteria along an externally applied magnetic field, certain characteristics of Magnetospirillum sp. AMB-1 cells were examined, including their growth pattern, motility, magnetosensitivity, swimming speed, and cell length distribution. In addition, the effect of toxic compounds on the swimming speed was assessed relative to application as a toxicity sensor. With an inoculum of 1.0 x 10(8) cells/mL, the cells reached the stationary phase with a concentration of about 5 x 10(8) cells/mL after 20 h, under both aerobic and anaerobic conditions. The distribution of the cell length did not vary significantly during the growth period, and both aerobically and anaerobically growing cells showed a similar cell length distribution. Although the cells showed similar growth patterns under both conditions, the anaerobically grown cells exhibited higher motility and magnetosensitivity. Actively growing cells under anaerobic conditions had an average swimming speed of 49 microm/s with a standard deviation of 20 microm/s. When the anaerobically growing cells were exposed to various concentrations of toxic compounds, such as 1-propanol and acetone, the swimming speed decreased with an increased concentration of the toxic compound. Accordingly, the relationship between swimming speed and toxicity can be used as an effective quantitative toxicity measurement; furthermore, the relative sensitivity of the proposed system was comparable to Microtox, which is commercially available.

Bacteriophage λ containing a cloned-gene is stably maintained in Escherichia coli in the lysogenic state while it is replicated and it overproduces a recombinant protein product in the lytic state. The host cell is eventually lysed in the lytic state. The kinetics of cell lysis and production induction were studied and are reported in this article through model equations. In two-stage continuous operation, the first tank is maintained in the lysogenic state for cell growth and cloned-gene stability while the second tank is in the lytic state for the overproduction of cloned-gene product. Individual cells in the second tank have different extent of the induction for product formation, since each has a different residence time. The different residence time for individual cells was taken into account using a population model. The numerical results show good agreement with the experimental data for the prediction of dilution rate in the second tank which gives the maximum product concentration.

The effect of silkworm hemolymph on baculovirus-induced insect cell apoptosis was investigated. The addition of silkworm hemolymph into the culture medium either before or during the baculovirus infection increased the host cell longevity; however, its addition after the infection was less effective. This can be explained by the higher transfer rate of silkworm hemolymph which is caused by endocytosis during the virus internalization step. The delayed cell death due to silkworm hemolymph was not caused by an inhibition of the virus attachment and internalization steps. The apoptosis was analyzed using DNA fragmentation and TUNEL assays, and the resulting data confirm that silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis.

In high density cultivation of Spodoptera frugiperda (Sf9) cells in Grace's medium supplemented with FBS (fetal bovine serum) and yeastolate, amino acids were the primary limiting substrates while the carbon sources were not. Glutamine, methionine, and threonine were consumed rapidly during the cultivation. When cultures were supplemented with amino acids, yeastolate components other than amino acids became the secondary limiting substrates.

Enterobacter cloacae is a facultative anaerobic bacterium to be an important cause of nosocomial infection. However, the isolated E. cloacae GGT036 showed higher furfural-tolerant cellular growth, compared to industrial relevant strains such as Escherichia coli and Corynebacterium glutamicum. Here, we report the complete genome sequence of E. cloacae GGT036 isolated from Mt. Gwanak, Seoul, Republic of Korea. The genomic DNA sequence of E. cloacae GGT036 will provide valuable genetic resources for engineering of industrially relevant strains being tolerant to cellular inhibitors present in lignocellulosic hydrolysates.

Hemolymph is oxidized and darkens visibly during the collection from silkworms due to the activity of tyrosinase in it. Toxic quinones are produced by the oxidation and consequently inhibit the cell growth. Heat treatment can be used to prevent the oxidation; however, the oxidation may occur during the collection of hemolymph before it is heat-treated. It makes the hemolymph collection difficult especially on a large-scale preparation. Hemolymphs collected from 257 different strains of silkworms were examined to select the slowly oxidized hemolymphs. Hemolymphs collected from mutant strains such as Lemone, TBO, Cre, Y4, and wEb showed relatively slow color changes. Oxidation rates of the hemolymphs were measured by the absorbance change using a spectrophotometer. The hemolymph of wEb showed the slowest oxidation. The absorbance of this mutant hemolymph reached the saturation value at 20°C in 450 min, whereas the total oxidation time of the wild-type (Baekokjam) hemolymph at the same temperature was 120 min. We tested if this mutant hemolymph is useful as a medium supplement for insect cell culture. Cell growth rate and final cell concentration in the medium supplemented with the wEb hemolymph were almost same as those in the medium supplemented with the wild-type hemolymph. Hemolymph is collected on a small scale by clipping the abdominal leg; however, this method is not appropriate for large scale preparation. Centrifugation after chopping the silkworm hemolymph by a blending mixer is a more appropriate procedure for large scale collection. Slowly oxidized wEb hemolymph resulted in higher cell concentration than the wild-type hemolymph when hemolymph was collected by the large scale preparation method.

We previously demonstrated that the lambda system integrated into the host chromosome can overcome the instability encountered in continuous operations of unstable plasmid-based expression vectors. High stability of a cloned gene in a lysogenic state and a high copy number in a lytic state provide cloned-gene stability and overexpression in a two-stage continuous operation. But the expression by the commonly used S- mutant lambda was only twice as high as that of the single copy. To increase the expression in the lambda system, we constructed a Q- mutant lambda vector that can be used in long-term operations such as a two-stage continuous operation. The Q- mutant phage lambda is deficient in the synthesis of proteins involved in cell lysis and lambda DNA packaging, while the S- mutant is deficient in the synthesis of one of two phage proteins required for lysis of the host cell and liberation of the progeny phage. Therefore, it is expected that the replicated Q- lambda DNA containing a cloned gene would not be coated by a phage head and would remain naked for ample expression of the cloned gene and host cells would not lyse easily and consequently would produce larger amounts of cloned-gene products. The beta-galactosidase expression per unit cell by the Q- mutant in a lytic state was about 30 times higher than that in a lysogenic state, while the expression by the commonly used S- mutant in a lytic state was twice as high as that in a lysogenic state. The optimal switching time of the Q- mutant from the lysogenic state to the lytic state for the maximum production of beta-galactosidase was 5.3 h, which corresponds to an early log phase in the batch operation.

The development of molecular detection that allows rapid responses with high sensitivity and selectivity remains challenging. Herein, we demonstrate the strategy of novel bio-nanotechnology to successfully fabricate high-performance dopamine (DA) biosensor using DA Receptor-containing uniform-particle-shaped Nanovesicles-immobilized Carboxylated poly(3,4-ethylenedioxythiophene) (CPEDOT) NTs (DRNCNs). DA molecules are commonly associated with serious diseases, such as Parkinson's and Alzheimer's diseases. For the first time, nanovesicles containing a human DA receptor D1 (hDRD1) were successfully constructed from HEK-293 cells, stably expressing hDRD1. The nanovesicles containing hDRD1 as gate-potential modulator on the conducting polymer (CP) nanomaterial transistors provided high-performance responses to DA molecule owing to their uniform, monodispersive morphologies and outstanding discrimination ability. Specifically, the DRNCNs were integrated into a liquid-ion gated field-effect transistor (FET) system via immobilization and attachment processes, leading to high sensitivity and excellent selectivity toward DA in liquid state. Unprecedentedly, the minimum detectable level (MDL) from the field-induced DA responses was as low as 10 pM in real- time, which is 10 times more sensitive than that of previously reported CP based-DA biosensors. Moreover, the FET-type DRNCN biosensor had a rapid response time (

The development of a cell-based high-throughput screening system has attracted much attention from researchers who study drug screening mechanisms and characterization of G-protein coupled receptors (GPCRs). Although olfactory receptors (ORs) constitute the largest group of GPCRs that play a critical role recognizing and discriminating odorants, only a few ORs have been characterized, and most remain orphan. The conventional cell-based assay system for characterizing GPCRs, including ORs, is very laborious, time consuming, and requires an expensive assay system. In this study, we developed a simple, low-cost miniaturized odorant screening method by combining Micro-Electro-Mechanical system (MEMs) technique and visualization technique for detecting an odorant response. We fabricated PEG microwell from a photocrosslinkable polyethylene glycol diacrylate (PEGDA) solution and applied it to cell culture and a reverse transfection platform for cell-based high-throughput screening. For the first time, the olfactory receptors were expressed on the microwell platform using reverse transfection technique. The various olfactory receptors can be expressed simultaneously using this technique and the microwell spotted with olfactory receptor genes can be used as a high-throughput screening platform. The odorant response was detected via fluorescence analysis on the microwell using a cAMP response element (CRE) reporter assay. We tested this platform using four de-orphaned ORs. This new cell-based screening method not only reduced numerous time-consuming steps but also allowed for simple, efficient, and quantitative screening and patterning of large numbers of GPCRs including ORs, which can help to visualize the OR response to odorants on a microwell.

An obligately aerobic, chemoheterotrophic, mesophilic prosthecate bacterium, designated strain CGM1-3ENT, was isolated from the enrichment cultures of forest soil from Cheonggyesan Mountain, Republic of Korea. Cells were Gram-reaction-negative, motile rods (1.3–2.4 µm long by 0.30–0.75 µm wide) with single flagella. The strain grew at 10–37 °C (optimum 25–30 °C) and at pH 4.5–9.5 (optimum 5.0–7.0). The major cellular fatty acids were C16 : 0, C18 : 1ω7c 11-methyl, C12 : 1 3-OH and summed feature 8 (comprising C18 : 1ω7c/C18 : 1ω6c). The genomic DNA G+C content of strain CGM1-3ENT was 63.7 mol%. The closest phylogenetic neighbour to strain CGM1-3ENT was identified as Asticcacaulis biprosthecium DSM 4723T (97.2 % 16S rRNA gene sequence similarity) and the DNA–DNA hybridization value between strain CGM1-3ENT and A. biprosthecium DSM 4723T was less than 24.5 %. Strain CGM1-3ENT used d-glucose, d-fructose, sucrose, maltose, trehalose, d-mannose, d-mannitol, d-sorbitol, d-galactose, cellobiose, lactose, raffinose, fumarate, pyruvate, dl-alanine and glycerol as carbon sources. Based on data from the present polyphasic study, the forest soil isolate CGM1-3ENT is considered to represent a novel species of the genus Asticcacaulis , for which the name Asticcacaulis solisilvae sp. nov. is proposed. The type strain is CGM1-3ENT ( = AIM0088T = KCTC 32102T = JCM 18544T).

Human alpha-galactosidase A (GLA) has been used in enzyme replacement therapy for patients with Fabry disease. We expressed recombinant GLA from Chinese hamster ovary cells with very high productivity. When compared to an approved GLA (agalsidase beta), its size and charge were found to be smaller and more neutral. These differences resulted from the lack of terminal sialic acids playing essential roles in the serum half-life and proper tissue targeting. Because a simple sialylation reaction was not enough to increase the sialic acid content, a combined reaction using galactosyltransferase, sialyltransferase, and their sugar substrates at the same time was developed and optimized to reduce the incubation time. The product generated by this reaction had nearly the same size, isoelectric points, and sialic acid content as agalsidase beta. Furthermore, it had better in vivo efficacy to degrade the accumulated globotriaosylceramide in target organs of Fabry mice compared to an unmodified version. [BMB Reports 2013; 46(3): 157-162]

The importance of creating a three-dimensional (3-D) multicellular spheroid has recently been gaining attention due to the limitations of monolayer cell culture to precisely mimic in vivo structure and cellular interactions. Due to this emerging interest, researchers have utilized new tools, such as microfluidic devices, that allow high-throughput and precise size control to produce multicellular spheroids. We have developed a droplet-based microfluidic system that can encapsulate both cells and magnetic nanoparticles within alginate beads to mimic the function of a multicellular tumor spheroid. Cells were entrapped within the alginate beads along with magnetic nanoparticles, and the beads of a relatively uniform size (diameters of 85% of the beads were 170-190 μm) were formed in the oil phase. These beads were passed through parallel streamlines of oil and culture medium, where the beads were magnetically transferred into the medium phase from the oil phase using an external magnetic force. This microfluidic chip eliminates additional steps for collecting the spheroids from the oil phase and transferring them to culture medium. Ultimately, the overall spheroid formation process can be achieved on a single microchip.

An exponential feeding strategy has been frequently used in fed-batch fermentation of recombinantE. coli. In this feeding scheme, growth yield and initial cell concentration, which can be erroneously determined, are needed to calculate the feed rate for controlling specific growth rate at the set point. The effect of the incorrect growth yield and initial cell concentration on the control of the specific growth rate was theoretically analyzed. Insignificance of the correctness of those parameters for the control of the specific growth rate was shown theoretically and experimentally.

Overexpression of bovine somatotropin (bST) results in the formation of inclusion bodies, leading to abnormal elongation of the Escherichia coli cell. As the cell elongates, the fraction of the inclusion body in the cell decreases and, consequently, bST productivity is lowered. Delayed expression of bST can be expected to prevent cell elongation; however, the trp promoter is leaky and incompletely repressed by tryptophan. The addition of 15 g/ l yeast extract, which contained various aromatic amino acids, into the starting medium delayed the derepression of the trp promoter significantly, whereas the addition of 100 mg/ l tryptophan did not. With the delayed expression, no cell elongation was observed until harvest and the final bST productivity increased more than three-fold. Intracellular tryptophanase, which degrades tryptophan, is another factor influencing regulation of the trp promoter. Expression of the tryptophanase operon is known to be subject to catabolite repression. The addition of a monosaccharide such as glucose, fructose, or galactose into the seed culture medium delayed the derepression of the trp promoter further in the main fed-batch fermentation.

Two-phase cultivation of insect cells was carried out to produce recombinant β-galactosidase and evaluate factors influencing high cell density and high expression. The primary factor limiting specific expression at high cell concentration was not the cell concentration but the medium condition. The addition of 4 g/L yeastolate during the growth phase increased cell density while the addition of 0.6 g/L glutamine in the production phase raised the specific expression rate. Productivity of recombinant protein was consequently improved seven-fold by this operation.