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Arginine deiminase (ADI) is a microbial-derived enzyme which catalyzes the conversion of L-arginine into L-citrulline. ADI originating from Mycoplasma has been reported to present anti-tumor activity against arginine-auxotrophic tumors, including melanoma. Melanoma cells are sensitive to arginine depletion due to reduced expression of argininosuccinate synthase 1 (ASS1), a key enzyme for arginine biosynthesis. However, clinical applications of recombinant ADI for melanoma treatment present some limitations. Since recombinant ADI is not human-derived, it shows instability, proteolytic degradation, and antigenicity in human serum. In addition, there is a problem of drug resistance issue due to the intracellular expression of once-silenced ASS1. Moreover, recombinant ADI proteins are mainly expressed as inclusion body forms in Escherichia coli and require a time-consuming refolding process to turn them back into active form. Herein, we propose fusion of recombinant ADI from Mycoplasma hominis and 30Kc19α, a cell-penetrating protein which also increases stability and soluble expression of cargo proteins, to overcome these problems. We inserted matrix metalloproteinase-2 cleavable linker between ADI and 30Kc19α to increase enzyme activity in melanoma cells. Compared to ADI, ADI-LK-30Kc19α showed enhanced solubility, stability, and cell penetration. The fusion protein demonstrated selective cytotoxicity and reduced drug resistance in melanoma cells, thus would be a promising strategy for the improved efficacy in melanoma treatment. KEY POINTS: • Fusion of ADI with 30Kc19α enhances soluble expression and productivity of recombinant ADI in E. coli • 30Kc19α protects ADI from the proteolytic degradation by shielding effect, helping ADI to remain active • Intracellular delivery of ADI by 30Kc19α overcomes ADI resistance in melanoma cells by degrading intracellularly expressed arginine.

It is known that a dog possesses much better sense of smell than a human. It has been reported that a dog has advantages (compared to a human) in the number of olfactory receptor (OR) genes, proteins and cells, and also nasal structure. However, a definitive reason for superior sensitivity of the canine olfactory system remains controversial. In this study, we compared sensitivity of human and canine olfactory receptors relative to the same condition. Human OR (hOR2W1) and canine OR (cfOR0312), previously identified to recognize hexanal, were inserted to the pcDNA3 vector. This vector was transfected to HEK293 cells. Hexanal-discriminating ability of ORs was confirmed using Fura-2 AM, a dye which illuminates when binding to calcium ions flowing into cells once olfactory signaling occurs. Consequently, cfOR0312 was more sensitive to hexanal than hOR2W1. Comparison of OR sensitivity would be one of the major factors in clarifying difference of sensitivity between human and canine olfactory systems.

We demonstrate the mechanism by which C3G, a major dietary anthocyanin, regulates energy metabolism and insulin sensitivity. Oral administration of C3G reduced hepatic and plasma triglyceride levels, adiposity, and improved glucose tolerance in mice fed high-fat diet. Hepatic metabolomic analysis revealed that C3G shifted metabolite profiles towards fatty acid oxidation and ketogenesis. C3G increased glucose uptake in HepG2 cells and C2C12 myotubes and induced the rate of hepatic fatty acid oxidation. C3G directly interacted with and activated PPARs, with the highest affinity for PPARα. The ability of C3G to reduce plasma and hepatic triglycerides, glucose tolerance, and adiposity and to induce oxygen consumption and energy expenditure was abrogated in PPARα-deficient mice, suggesting that PPARα is the major target for C3G. These findings demonstrate that the dietary anthocyanin C3G activates PPARs, a master regulators of energy metabolism. C3G is an agonistic ligand of PPARs and stimulates fuel preference to fat., Jia, Wu, Kim et al. show that cyanidin-3-O-glucoside (C3G), a major dietary anthocyanin, functions as a ligand for PPARalpha to regulate energy metabolism and insulin sensitivity in mouse models of obesity and diabetes. This study suggests that C3G slows down the metabolism of glucose, making it a promising therapeutic agent.

A bioelectronic nose device based on micelle-stabilized olfactory receptors is developed for the selective discrimination of a butter flavor substance in commercial fermented alcoholic beverages. In this work, we have successfully overexpressed ODR-10, a type of olfactory receptor, from Caenorhabditis elegans using a bacterial expression system at a low cost and high productivity. The highly-purified ODR-10 was stabilized in micelle structures, and it was immobilized on a carbon nanotube field-effect transistor to build a bioelectronic nose for the detection of diacetyl, a butter flavor substance, via the specific interaction between diacetyl and ODR-10. The bioelectronic nose device can sensitively detect diacetyl down to 10 fM, and selectively discriminate it from other substances. In addition, this sensor could directly evaluate diacetyl levels in a variety of real fermented alcoholic beverages such as beer, wine, and makgeolli (fermented Korean wine), while the sensor did not respond to soju (Korean style liquor without diacetyl). In this respect, our sensor should be a powerful tool for versatile food industrial applications such as the quality control of alcoholic beverages and foods.

Hormones within very low levels regulate and control the activity of specific cells and organs of the human body. Hormone imbalance can cause many diseases. Therefore, hormone detection tools have been developed, particularly over the last decade. Peptide hormones have a short half-life, so it is important to detect them within a short time. In this study, we report two types of peptide hormone sensors using human hormone receptor-carrying nanovesicles and graphene field-effect transistors (FETs). Parathyroid hormone (PTH) and glucagon (GCG) are peptide hormones present in human blood that act as ligands to G protein-coupled receptors (GPCRs). In this paper, the parathyroid hormone receptor (PTHR) and the glucagon receptor (GCGR) were expressed in human embryonic kidney-293 (HEK-293) cells, and were constructed as nanovesicles carrying the respective receptors. They were then immobilized onto graphene-based FETs. The two hormone sensors developed were able to detect each target hormone with high sensitivity (ca. 100 fM of PTH and 1 pM of GCG). Also, the sensors accurately recognized target hormones among different types of peptide hormones. In the development of hormone detection tools, this approach, using human hormone receptor-carrying nanovesicles and graphene FETs, offers the possibility of detecting very low concentrations of hormones in real-time.

The curry compound, curcumin exerts multiple health-promotive functions; however, its poor solubility and stability limits its biological applications. In this study, we illuminate intermolecular binding mechanisms in the nano-sized complex of curcumin with silkworm protein, 30Kc19. The intrinsic fluorescence of 30Kc19 was gradually quenched by the increase of curcumin concentrations, which demonstrates molecule-molecule complexations mediated by the fluorophore amino acid residues (Tyr, Trp) in the protein. The fluorescence quenching showed that the binding occurred at 1:1 molar ratio with binding constant of 3.28 × 104 M-1. The results from scanning electron microscopy and dynamic light scattering indicate that the complexes were formed with cubicle shapes and sizes of 200–250 nm at pH 8.0 (zeta-potential < −20 mV). Along with Fourier transform infrared analysis, computational studies of protein-ligand docking simulation suggest a mechanism that curcumin and 30Kc19 forms complexes through specific amino acid residues (Trp174, Trp180, and Trp225) with minimum binding distance (4 A). The complexation of curcumin with 30Kc19 protein effectively suppressed the degradation of curcumin over 10 h and improved its antioxidant activity up to 30%. These findings suggest an application of 30Kc19 for the delivery of waterinsoluble bioactive medicines.

Biomass contains cellulose, xylan and lignin in a complex interwoven structure that hinders enzymatic hydrolysis of the cellulose. To separate these components in yellow poplar biomass, we sequentially pretreated with dilute sulfuric acid and enzymatically-generated peracetic acid. In the first step, the dilute acid with microwave heating (140°C, 5 min) hydrolyzed 90% of xylan. The xylose yield in hydrolysate after dilute acid pretreatment was 83.1%. In the second step, peracetic acid (60°C, 6 h) removed up to 80% of lignin. This sequential pretreatment fractionated biomass into xylan and lignin, leaving a solid residue enriched in cellulose (~80%). The sequential pretreatment enhanced enzymatic digestibility of the cellulase by removal of the other components in biomass. The glucose yield after enzymatic hydrolysis was 90.5% at a low cellulase loading (5 FPU/g of glucan), which is 1.6 and 18 times higher than for dilute acid-pretreated biomass and raw biomass, respectively. This novel sequential pretreatment with dilute acid and peracetic acid efficiently separates the three major components of yellow poplar biomass, and reduces the amount of cellulase needed.

Ultraviolet (UV) irradiation induces skin photoaging by generating reactive oxygen species (ROS). ROS caused by UV-irradiation results in loss of skin cells and degradation of extracellular matrix. A number of antioxidants have been chemically synthesized or naturally extracted to prevent ROS-mediated skin photoaging. In our previous work, silkworm hemolymph extract (SHEX) was prepared, and its antioxidant activity was tested by free radical-scavenging assay. This study assessed the protective effects of SHEX on UV-induced photoaging of human immortalized keratinocytes (HaCaT). UVA (365 nm)-induced ROS generation was inhibited by supplementation of silkworm hemolymph (SH). Treatment with SHEX prepared by boiling SH inhibited death of HaCaT cells caused by UVB (315 nm) and UVA irradiation in a dose-dependent manner. Seven fractions were obtained by separating SHEX by gel permeation chromatography and the antioxidant activity of the fractions was examined. The fraction showing the highest protective efficacy on UV-induced cell damage corresponded to the lutein-containing fraction isolated in our previous study. Moreover, the SHEX fraction suppressed the expression of MMP-1 (matrix metalloproteinase-1), a matrix-degrading enzyme, suggesting that the active constituent of SHEX has the potential to inhibit skin photoaging. These results suggest that SHEX can be developed as a dietary and cosmetic supplement for prevention of skin photoaging.

We report a strategy for the human-like smelling of a rose scent utilizing olfactory receptor nanodisc (ND)-based bioelectronic nose devices. In this strategy, a floating electrode (FE)-based carbon nanotube (CNT) field effect transistor (FET) was functionalized with human olfactory receptor 1A2 (hOR1A2)-embedded NDs (hOR1A2NDs). The hOR1A2NDs responded to rose scent molecules specifically, which were monitored electrically using the underlying CNT-FET. This strategy allowed us to quantitatively assess the contents of geraniol and citronellol, the main components of a rose scent, as low as 1 fM and 10 fM, respectively. In addition, it enabled us to selectively discriminate a specific rose odorant from other odorants. Significantly, we also demonstrated that the responses of hOR1A2NDs to a rose scent could be strongly enhanced by enhancer materials like a human nose. Furthermore, the method provided a means to quantitatively evaluate rose scent components in real samples such as rose oil. Since our method allows one to quantitatively evaluate general rose scent ingredients just like a human nose, it could be a powerful strategy for versatile basic research and various applications such as fragrance development.

Silkworm contains several distinct proteins that have anti-apoptotic activities in mammalian and insect cells. Although the characteristics of 30K proteins have been well investigated, those of SPs (Storage Proteins) have not been examined. In this article, an anti-apoptotic effect of SP1 was investigated in HeLa cell apoptosis. SP1, a type of insect hemocyanin protein, is a female-specific protein in silkworm and consists of high portion of methionine. Transient expression of SP1 in HeLa cells slightly increased cell viability after the treatment with staurosporine, which induced apoptosis via intrinsic mitochondrial pathway. To investigate the anti-apoptotic effect of SP1, the stable cell line expressing SP1 were screened and constructed. The anti-apoptotic effect of the cell line was assessed using trypan blue exclusion assay, microscopic analysis of nuclear fragmentation, and flow cytometry. SP1 expressing cell line showed strong antiapoptotic activity against the treatment of staurosporine but not TNFa, meaning that SP1 is a protein inhibitor of apoptosis caused by intrinsic apoptosis pathway. As the SP1 is the novel anti-apoptotic protein, the regulation of apoptosis by SP1 will offer a new strategy that can be utilized in biopharmaceutical industry when the focus is on minimization of cell death and maximization of productivity.

Olfactory receptors (ORs), belonging to the Gprotein coupled receptor (GPCR) family, are very difficult to be overexpressed, purified and reconstituted because of their hydrophobicity and complicated structure. These receptors bind to their specific ligands, thus their specificity is very useful for application as a bioelectronic nose. Furthermore, highly purified and well-reconstituted human olfactory receptor (hOR) can be used in various fields, such as in protein-interaction research, drug screening, and analysis of the hOR structure. In this study, human olfactory receptor, hOR2AG1, was produced with high purity and functionally reconstituted in detergent micelles. The hOR2AG1 was overexpressed in Escherichia coli (E. coli) with glutathione S-transferase (GST) and 6xHis-tag as an inclusion body. The hOR2AG1 fusion protein was solubilized in buffer containing sodium dodecyl sulfate (SDS) and purified using Ni-NTA chromatography. The GST domain was removed using proteolytic cleavage before elution from the column. After purification, the hOR2AG1 was successfully reconstituted using nonionic detergents and methyl-s-cyclodextrin. Finally highly purified and well-reconstituted hOR was obtained, and its biological characteristics were confirmed by using circular dichroism (CD) spectrum and tryptophan fluorescence assay. These results can be applied to develop protein-based sensing systems including a bioelectronic nose and to analyze the native hOR structure using solid-state NMR, X-ray crystallography, or neutron scattering.

We investigated the effect of light intensity on the correlation between optical density (OD) and cell mass concentration in high cell density culture of Anabaena variabilis. When the light intensity was gradually increased to maintain specific irradiation rate above 10 mmol/s/g dry cell, the dry cell mass concentration to OD ratio changed when the cell density became higher than OD 10, while the correlation was linear when light intensity remained constant after 2.5 days. When the OD was below 10, the dry cell concentration to OD ratio in unicells was the same as that of filaments, indicating that filament length, unicell size, and unicell dry cell concentration did not affect the dry cell concentration to OD ratio. Scanning electron microscope pictures revealed the differences in morphological structures between filaments below and above OD 10. Therefore, we propose that the morphological structures of filaments affected the dry cell concentration to OD ratio.

Pretreatment of biomass with dilute acid requires high temperatures of >160 °C to remove xylan and does not remove lignin. Here we report that the addition of peracetic acid, a strong oxidant, to mild dilute acid pretreatment reduces the temperature requirement to only 120 °C. Pretreatment of yellow poplar with peracetic acid (300 mM, 2.3 wt%) and dilute sulfuric acid (100 mM, 1.0 wt%) at 120 °C for 5 min removed 85.7% of the xylan and 90.4% of the lignin leaving a solid consisting of 75.6% glucan, 6.0% xylan and 4.7% lignin. Low enzyme loadings of 5 FPU/g glucan and 10 pNPGU/g glucan converted this solid to glucose with an 84.0% yield. This amount of glucose was 2.5 times higher than with dilute acid-pretreated solid and 13.8 times higher than with untreated yellow poplar. Thus, the addition of peracetic acid, easily generated from acetic acid and hydrogen peroxide, dramatically increases the effectiveness of dilute acid pretreatment of biomass.

Nanomaterials are extensively used in consumer products and medical applications, but little is known about their environmental and biological toxicities. Moreover, the toxicity analysis requires sophisticated instruments and labor-intensive experiments. Here we report a microfluidic chip incorporated with the nematode Caenorhabditis elegans that rapidly displays the changes in body growth and gene expression specifically responsive to the silver nanoparticles (AgNPs). C. elegans were cultured in microfluidic chambers in the presence or absence of AgNPs and were consequently transferred to wedge-shaped channels, which immobilized the animals, allowing the evaluation of parameters such as length, moving distance, and fluorescence from the reporter gene. The AgNPs reduced the length of C. elegans body, which was easily identified in the channel of chip. In addition, the decrease of body width enabled the worm to advance the longer distance compared to the animal without nanoparticles in a wedge-shaped channel. The transgenic marker DNA, mtl-2::gfp was highly expressed upon the uptake of AgNPs, resulting in green fluorescence emission. The comparative investigation using gold nanoparticles and heavy-metal ions indicated that these parameters are specific to AgNPs. These results demonstrate that C. elegans-on-a-chip has a great potential as a rapid and specific nanoparticle detection or nanotoxicity assessment system.

Macrophages participate in several inflammatory pathologies such as sepsis and arthritis. We investigated the effect of silkworm hemolymph (SH) on the LPS-induced pro-inflammatory macrophages. SH inhibits LPS-induced nitric oxide (NO) production in RAW 264.7 cells and murine peritoneal macrophages. The decreased NO was reflected as a decreased amount of inducible nitric oxide synthase (iNOS) mRNA and protein. It was also found that SH inhibited pro-inflammatory cytokines, IL-1β, IL-6, and TNF-α production. To elucidate the mechanism by which SH inhibits NO production and iNOS expression, we investigated that SH suppressed IκB phosphorylation, which leads to the activation of NF-κB followed by degradation of IκB. This observation suggests that SH is a potential therapeutic modulator for inflammation-associated disorders.

Molecular computing using programmable nucleic acids has been attracting attention for use in autonomous sensing systems and information processing systems by interacting with a biological environment. Here, we introduce a rule-based in vitro molecular classification system that can classify disease patterns using several microRNA (miRNA) markers via the assembly of programmed DNA strands. The classification rules were derived by analyzing large-scale miRNA expression data obtained from a public database, and the identified rules were converted into DNA sequences. Classification was performed via the detection of miRNA markers in the rules. The classification results were reported as a binary output pattern according to their hybridization to the rule sequences, which can be conveniently visualized using gold nanoparticle aggregation. Our results demonstrate the utility of in vitro molecular classification by illustrating one of the ways in which molecular computing can be used in future biological and medical applications.

Silkworm was used in the sericulture industry and also processed for food sources in some of Asian countries. In previous studies, it was reported that the 30K protein in silkworm, Bombyx mori hemolymph inhibited animal cell apoptosis. In this work, the antioxidative function of silkworm hemolymph was investigated using its protein-free extract. H2O2-induced ROS and O2·− were efficiently scavenged in Chinese hamster ovary cells when the culture medium was supplemented with silkworm hemolymph. The hemolymph extract prepared by deproteinization through ethanol precipitation completely scavenged DPPH radicals (IC50=0.025 mg/mL). When the extract was supplemented with protein-free medium, oxidative damage to mitochondria was efficiently diminished with the maintenance of the mitochondrial membrane potential. Lutein was identified as the major antioxidative constituents in the extract (52.06 μg/mg).

We previously reported that the expression of Bombyx mori 30Kc19 gene in CHO cells significantly improved both the production and sialylation of recombinant human EPO (rHuEPO) in adhesion culture mode. In this study, the effects of 30Kc19 expression and supplementation of 30Kc19 recombinant protein on the productivity and glycosylation pattern of rHuEPO were investigated in the serum-free suspension culture mode. Especially, glycosylation pattern was examined in detail using a quantitative MALDI-TOF MS method. The expression of 30Kc19 increased the EPO production by 2.5-folds and the host cells produced rHuEPO with more complex glycan structures and a larger content of sialic acid and fucose. The glycan structures of rHuEPO in the 30Kc19-expressing cell consisted of bi-, tri-, tetra-, and penta-antennary branching (35, 18, 33, and 14 %, respectively), while the control cells produced predominantly bi-antennary branching (70 %). About 53 % of the glycans from rHuEPO in the 30Kc19-expressing cell was terminally sialylated, while no obvious sialylated glycan was found in the control cells. The percentage of fucosylated glycans from the 30Kc19-expressing cell culture was 77 %, whereas only 61 % of the glycans from the control cell were fucosylated glycans. We also examined whether these effects were observed when the recombinant 30Kc19 protein produced from Escherichia coli was supplemented into the culture medium for CHO cells. In the control cell line without the 30Kc19 gene, EPO production increased by 41.6 % after the addition of 0.2 mg/mL of the recombinant 30Kc19 protein to the culture medium. By the Western blot analysis after two-dimensional electrophoresis (2-DE) of isoforms of EPO, we confirmed that 30Kc19 enhanced the sialylation of EPO glycans. These results demonstrated that both 30Kc19 gene expression and the recombinant 30Kc19 protein addition enhanced rHuEPO productivity and glycosylation in suspension culture. In conclusion, the utilization of 30Kc19 in CHO cell culture holds great promise for use in the manufacturing of improved biopharmaceutical glycoproteins.

Previously, we reported that the expression of Bombyx mori 30Kc6 gene in Chinese hamster ovary (CHO) cells increases recombinant protein production by both inhibiting apoptosis and enhancing specific productivity. In this study, in order to gain a thorough understanding of the roles of 30Kc6 gene in antibody production, the mechanisms modulating cell apoptosis and specific productivity were investigated. 30Kc6 gene was introduced into a CHO cell line producing a chimeric anti-human CD20 monoclonal antibody. The stable expression of 30Kc6 increased cell viability and productivity by 46.7% and 3.4-folds, respectively. It was observed that the Bax translocation from cytosol to mitochondria and the cytochrome c (cyt c) release from mitochondrial intermembrane space to cytosol were repressed, which resulted in a decrease in the activation of apoptosis executioner, caspase-3. On the other hand, 30Kc6 expression increased the specific productivity by 2.3-folds. However, at the transcription level, the relative levels of heavy and light chain mRNAs increased only by 8.3% and 8.7%, respectively, which was not accountable for the observed increment in the specific productivity. Instead, the mitochondrial membrane potential was maintained and the ATP generation was stimulated. A higher ATP level could activate the mammalian target of rapamycin (mTOR), which drives the translation initiation and elongation by phosphorylating eukaryotic initiation factor 4E binding protein 1 (4EBP1) and S6 kinase 1 (S6K1). In the 30Kc6-expressing cells, both the 4EBP1 and S6K1 were phosphorylated at higher levels, which indicated that the increased specific productivity primarily resulted from the boost of translation process. Furthermore, it was also found that the specific uptake rates of glucose and glutamine were not affected by 30Kc6 expression, demonstrating that the enhanced ATP generation and consequently maintained mTOR activity were due to 30Kc6 expression but not the different metabolic uptake rates. In conclusion, 30Kc6 expression inhibited apoptosis by repressing the Bax translocation, which down-regulated the downstream cascade responses including cyt c release and caspase-3 activation. Also, 30Kc6 expression increased the specific productivity by enhancing the translation process.

The market for protein-drugs has steadily increased due their increased use as alternatives to traditional small molecule drugs. While some therapeutic proteins have been produced in microbial systems, mammalian cell systems such as Chinese hamster ovary (CHO) cells are widely used as the host cell system. To increase the efficiency of producing therapeutic proteins, many researchers have attempted to solve the critical problems that occur in mammalian cell systems. As a result, several serum-free media and advanced culture methods have been developed, and protein productivity has increased considerably through the development of efficient selection methods. However, the prevalence of apoptosis during mammalian cell culture still remains a significant problem. Based on the understanding of apoptotic mechanisms and related proteins, anti-apoptotic engineering has steadily progressed. In this study, we review the strategies that have been developed for high-level production of recombinant proteins in the CHO cell system via a selection of clones, target-gene amplification, optimization of culture systems and an inhibition of apoptosis through genetic modification.

In this study, we used a bacteriophage λQ − S − mutant that increased the stability of recombinant Escherichia coli during continuous culture. The operation was conducted in two stages: the first stage was carried out to promote cell growth, and the second stage was performed for product formation. The productivity of recombinant proteins depends on the substrate concentration of the fresh medium supplied to the second stage (S 3) and dilution rate of the second stage (D 2). With the optimal value of S 3 and D 2, the first and second stages were stably maintained for 170 and 80 h, respectively. To further improve this process, a three-stage continuous process was conducted with an additional induction stage between the growth and production stages. Compared with the two-stage operation, the stable production period was extended by 1.7 fold, and the recombinant protein production increased by 1.3 fold.

The olfactory system has the ability to discriminate and identify thousands of odorant compounds at very low concentrations. Recently, many researchers have been trying to develop artificial sensing devices that are based on the olfactory system. A bioelectronic nose, which uses olfactory receptors (ORs) as sensing elements, would benefit naturally optimized molecular recognition. Accordingly, ORs can be effectively used as a biological element in bioelectronic noses. Bioelectronic nose can be classified into cell-based and protein-based biosensors. The cell-based biosensor uses living cells that express olfactory receptors as the biological sensing elements and the protein-based biosensor uses the olfactory receptor protein. The binding of odorant molecules to the ORs can be measured using various methods such as piezoelectric, optic, and electric devices. Thus, bioelectronic nose can be developed by combining the biological sensing elements with these non-biological devices. The application of bioelectronic nose in a wide range of different scientific and medical fields is essentially dependent on the development of highly sensitive and selective biosensors. These sensor systems for the rapid detection of specific odorants are crucial for environmental monitoring, anti-bioterrorism, disease diagnostics, and food safety. In this article, we reviewed recent advances in the development of bioelectronic nose.

The silkworm hemolymph has an anti-apoptotic activity in insect, mammalian, and human cell systems. The protein from silkworm hemolymph with the highest apoptosis inhibiting activity was found to be 30Kc19 protein, which was one of the ‘30K proteins’. In this study, 30Kc19 protein encoded by the 30Kc19 gene of the silkworm was expressed in Escherichia coli with (pET-22b(+)) and without (pET-3a) pelB leader sequence. 30Kc19 protein was over-expressed largely as a soluble form by pET-3a and both as soluble and insoluble forms by pET-22b(+). The medium was supplemented with each of the recombinant 30Kc19 proteins, and their presence was found to inhibit nuclear fragmentation and apoptotic body formation in actinomycin D-induced Sf9 cell apoptosis. Moreover, 30Kc19 protein repressed the activation of Sf-caspase-1. The 30Kc19 protein obtained from periplasm showed the most effective anti-apoptotic activity. This protein holds great potential for industrial and pharmaceutical applications since mass production and easy purification of this protein is possible.

Odorant-binding proteins (OBPs) primarily function in the transport of hydrophobic odorants. In this study, OBPs originating from rat and pig were cloned into a mammalian expression vector, pcDNA3, and expressed in HEK-293 cells, and their specificity for odorants and olfactory receptors was examined. Results suggest that OBPs have a high affinity for the olfactory receptors when both the OBP and receptor originate from the same species. The rat OBPs were bound not only to the rat olfactory receptor I7 but also to the odorant specific to I7. The solubility of the odorant was increased by both OBP2 and OBP3, which originate from rat, but with different efficiencies. These results demonstrate that OBPs specifically interact with odorants as well as olfactory receptors, and these interactions can influence the sensitivity of olfactory receptor-based biosensors.

Olfactory receptors pertaining to G protein-coupled receptor (GPCR) are integral membrane proteins composed of seven transmembrane spanning domains. It has been reported that these receptor proteins are difficult to overexpress, solubilize, and purify because of their complicated structures and strong hydrophobicity. In this study, full-length human olfactory receptor (hOR) 2AG1 was overexpressed in E. coli as a fusion protein with a glutathione S-transferase (GST) tag mainly as an inclusion body without any mutations or deletions in the gene. This protein was difficult to solubilize with detergents and chaotropic agents, and only N-lauroyl sarcosine was found to be suitable for solubilizing it. In contrast, Trition X-100 was found to solubilize most of the impurity proteins from the insoluble fraction in E. coli. Based on this observation, we applied a simple and efficient column-free method using these two detergents for the purification of the olfactory receptor protein. In this method, the insoluble fraction of the cell lysate was first treated with Triton X-100 to remove impurity proteins. The remaining insoluble fraction was then further treated with N-lauroyl sarcosine to solubilize the olfactory receptor protein. Milligram quantity of the human olfactory receptor was produced. This is the first report to produce full-length of the olfactory receptor from E. coli.

A common problem with the in vivo therapeutic applications of cells is that cells can rapidly disappear into the circulatory system after an injection. Magnetic nanoparticles can be used to solve this problem. Bacterial magnetic nanoparticles were used in this study for targeting stem cells at a specific location within a microfluidic channel. Magnetic nanoparticles were isolated from Magnetospirillum sp. AMB-1 and delivered to endothelial progenitor cells (EPCs). Cellular uptake of magnetic nanoparticles and their functional feasibility was characterized in vitro. The environment of a human blood vessel was simulated using a microfluidic channel. Magnetic nanoparticle-incorporated EPCs were injected into a microchannel and the flow rate of cells was uniformly controlled by use of a syringe pump. EPCs were effectively targeted to a specific location within the microchannel by an external magnetic field (about 400 mT). About 40% of EPCs were efficiently targeted with a flow rate of 5 microl min(-1) when 10 microg of magnetic nanoparticles were used per 10(4) cells. This microfluidic system provides a useful tool towards a better understanding of the behavior of magnetic nanoparticle-incorporated cells within the human circulatory system for clinical use.

In a two-phase operation, E. coli containing λSNNU1 (Q − S −) in the chromosome is typically cultured at 33°C and cloned gene expression is induced by elevating the temperature. At least 40°C is necessary for complete induction of cloned gene expression; however, temperatures above 40°C have been shown to inhibit cloned gene expression. This suggests that a three-phase operation, which has an induction phase between the growth and production phases, may result in higher gene expression. In this study, optimal temperature management strategies were investigated for the three-phase operation of cloned gene expression in thermally inducible E. coli/bacteriophage systems. The optimal temperature for the induction phase was determined to be 40°C. When the temperature of the production stage was 33°C, the optimal time period for the induction phase at 40°C was determined to be 60 min. In contrast, when the temperature of the production phase was 37°C, the optimal period for the induction phase at 40°C was 20∼30 min. When the three-phase temperature and temporal profile were set at a growth phase of 33°C, an induction phase at 40°C for 30 min, and a production phase at 37°C, the highest level of cloned gene expression was achieved.

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.

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.

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.

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.

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.

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.

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.

Optimum specific growth rate for the production of recombinant bovine somatotropin (bST) was investigated in fed-batchE. coli fermentation with a controlled specific growth rate. Maximum specific bST concentration based on cell mass decreases linearly with the controlled specific growth rate. Productivity of bST is expressed by a quadratic equation as a function of the specific growth rate and has a maximum value at μ=0.23 hr−1.

Tryptophan promoter is widely used in the industrial fermentation for the overproduction of recombinant proteins. It can be regulated by the concentrations of tryptophan and 3β-indolea-crylic acid (IAA). A mathematical model is proposed for the regulation of the promoter by the manipulation of tryptophan and IAA concentrations. The transcription initiation frequency increases with IAA concentration and reaches a plateau. The frequency decreases drastically even with the addition of a small amount of tryptophan. These behaviors agree with the previous experimental investigation.

The investigation of electroantennogram (EAG) using insect antennae has been primarily focused on the measurement of insect pheromone. Insect has highly specialized olfactory receptors inside their antennae. In this paper, EAG was applied to detect general odorants and the feasibility of this system for the olfactory biosensor was investigated. Electroantennogram measurement was carried out using the antennae of male silkworm moth,Bombyx mori, and ammonia gas as the model odorant. EAG parameters including peak amplitude, decay, and level were analyzed for the quantitative measurement. The peak amplitude increased linearly with the ammonia concentration and the reproducible electrical signals were generated at least for 2 hrs after the antenna was cut off from the silkworm moth.

The effectiveness of silkworm hemolymph was investigated as a substitute for fetal bovine serum (FBS) in insect cell culture. Cells were adapted to grow in reduced FBS medium supplemented with silkworm hemolymph through a gradual adaptation process. FBS concentration in the medium could be reduced to 1% without decrease in cell growth rate and maximum cell concentration by adding 5% silkworm hemolymph.

Growth limiting factors influencing the high density cultivation of insect cells were studied. A stationary phase in Grace's medium was found to be due to the deprivation of the active form of FBS components. Various compounds were added in the early stationary phase to observe the recovery of cell growth. With the addition of yeastolate, final cell densities were 4-fold and 3.5-fold higher in monolayer and suspension cultures, respectively. Pluronic F-68 increases the specific growth rate and the growth yield of the cell as well as protects the cell from the shear damage.

The effects of a derepressor, IAA (3β-indoleacrylic acid) ontrp promoter inE. coli are studied. Kinetic parameters for the effect are estimated through the model equations and experiments. IAA concentration of 40 μg/ml is high enough to give the maximum transcriptional efficiency in M9 media supplemented with glucose and casamino acid.

Silicone tube was used to facilitate gas transport in mammalian cell culture. A bundle of silicone tubes provides anchorage dependent ceils (BHK 21 cells) with surface. It was found that the surface characteristic of silicone tube for the cell attachment was the same as that of commercial tissue culture dish. Also high O2 permeability of silicone membrane gives rise to a faster metabolism of cells.

A novel bioreactor with hollow fibres was developed to facilitate substrate transfer across membrane walls as well as to retain a continuous cell growth in the shell side. Ultrafiltration was induced through membrane by pressurizing feed solution to the inside of a hollow fibre with inlet and outlet pumps. The ultrafiltrate accumulated outside the hollow fibres was recirculated through a reservoir where a part of solution containing cells and substrate was removed to keep the level of reservoir solution constant. Ethanol production by Saccharomyces cerevisiae was carried out to test the feasibility of this reactor. The productivity of this reactor was compared with that of a continuous stirred tank reactor (CSTR).