Earticle

We investigated the effects of Aurintricarboxylic Acid (ATA) on Chinese Hamaster Ovary (CHO) DG44 cell growth in serum free media with controlled calcium concentration, measuring the cell aggregation rate with the coulter counter. In previous studies, high levels of extracellular calcium ion (12.5 mg/L) showed to cause decreased cell density and high aggregation to CHO DG44 cells in serum-free media. Cell aggregation during cell culture causes hindrance of nutrient and oxygen transfer to the cell, leading to inhibition of cell growth. We observed increased cell growth and decreased cell aggregation when we added ATA in the calcium controlled medium. Exhibited effects of ATA on cell aggregation particularly appeared in high-calcium levels. In commercial serum-free media that cannot control calcium concentrations, ATA is a possible solution that may solve the cell aggregation problem in high-calcium conditions.

Cyclo-His-Pro (CHP) is a cyclic dipeptide with numerous biological activities, and structurally related to thyrotropin-releasing hormone that was originally discovered in brain. This study was designed to determine the protective effect and its action mechanism of CHP on the apoptosis of pancreatic β-cells. The RINm5F cell line is a well-established and commonly used in vitro model for the study of the biology of pancreatic islet cells. Hence, the effect of CHP was tested on streptozotocin (STZ)-treated RINm5F cells and their stimulatory effects on RINm5F cells proliferation and apoptosis were investigated. CHP was produced from defatted soybean meal by hydrolysis with commercial proteases. In RINm5F cells, CHP markedly protected against STZ-mediated cytotoxicity (76%). Moreover, RINm5F cells incubated with CHP showed a significant reductions of STZ-induced nitric oxide (NO) production and lipidperoxidation. It also reduced the STZ-induced apoptotic events such as the activation of caspase-3, poly (ADP-ribose) polymerase cleavage, and DNA fragmentation in RINm5F cells showing the mode of protection of CHP on RINm5F cells. In addition, RINm5F cells treated with CHP increased the insulin-secretion capacity. These results suggest that CHP may be a candidate for the therapeutic agent against STZ-mediated cytotoxicity and apoptosis.

Human cytotoxic T lymphocyte-associated antigen4-immunoglobulin (hCTLA4Ig), an immunosuppressive agent, was expressed in transgenic rice cells using RAmy3D promoter and RAmy1A signal peptide for the inducible production and secretion in to culture media by sugar depletion. When producing proteins by using transgenic plant cells, the biggest problem is low productivity. Since our system inducibly produces target proteins after the starvation of sugar, the production is possible only when there is no energy source. For this reason, the viability of the cells is reduced after induction. Therefore, this possibility of improving productivity by adding alternative energy source was investigated in this study. To improve the productivity of hCTLA4Ig, pyruvic acid and sodium pyruvate were added in transgenic rice cell cultures. When 87 and 174 mM pyruvic acid were added during the production phase, the viability of cells has been similar to that of control. However no hCTLA4Ig was produced. It has been shown that pyruvic acid was not an effective energy source in this system. Both concentrations (87 , 174 mM) of sodium pyruvate were also added to determine the optimal level. When 87 mM sodium pyruvate was added in suger-free media, the rate of cell death become lower and the production of hCTLA4Ig was increased. When sodium pyruvate were not added, viability was almost zero at day 6. However, by adding sodium pyruvate, 18% of viability could be kept until the tenth day. In addition, the production of hCTLA4Ig could be enhanced.

Porcine epidemic diarrhea virus (PEDV) is a Coronavirus and a causative agent for diarrhea in pigs, particularly in neonates. The propagation of the PEDV in cell culture was developed in 1988. Porcine epidemic diarrhea disease vaccine was produced from Vero cells by using lively attenuated virus strain[1]. The PEDV strain of PED-SM was tested in this study. The optimal MOI, infection time, and harvest time of PED-SM were 0.1, 72 hr of post-inoculation, and 24 hr of post-infection. With 0.1 MOI, PED-SM showed not only the highest virus production rate, but also highest cell death rate. Therefore, the maintenance of high cell density for long time is keenly needed for the high virus titer. A semi-perfusion culture of Vero cells was tried for the high cell density culture. The medium change with 10% FBS supplemented medium showed better cell growth than that with other concentration of FBS. However, the virus titer with medium change was lower than that with no medium change. This maybe caused by the depletion of nutrients in virus production medium due to the high cell density in semiperfusion culture. Accordingly, additional nutrients supplement maybe needed in the virus production step as well as in the cell culture step.

Factor X(FX) is a vitamin K-dependent plasma protease, two-chain glycoprotein that plays a central role in blood coagulation pathway. Factor X is activated to factor Xa (FXa) in vitro by a factor X activator isolated from Russell's viper venom (RVV). FXa cleaves after the arginine residue in its preferred cleavage site Ile-(Glu or Asp)-Gly-Arg, widely used to remove fusion tags from expressed proteins. In this study, expression was achieved using a full-length FX cDNA. Recombinant human factor X (hFX) was expressed in transgenic rice cell suspension culture under the control of rice amylase 3D promoter (pRAmy3D), which is induced by sugar starvation. hFX mRNA and protein expression in transgenic rice cells were detected by Northern and Western blot analyses. The proteolytic activity of factor Xa, hFX activated with RVV, was characterized by using a EGFP-hTNFa fusion protein as substrate, which contained a factor Xa cleavage site between two moieties. These results demonstrate that the hFX derived from rice cell suspension culture system can be used to cleave the tags from the fusion proteins (This Study was supported by a grant from the Next Generation New Technology Development Project (10030122) of the Ministry of Knowledge Economy).

Perfusion culture of mammalian cells including embryonic stem cell (ESC) is attractive, given that culture environment can be maintained consistently. Cell membrane motility deformed by fluidic stress presents aspects of adhesion, stretching and migration belonging to intercellular molecular dynamics. Consequently, cell feature and motility have been carried on cellular physicochemistry as key characteristic indices. We suggest Lf as a new parameter, which represents the free peripheral membrane length without attaching with other cells to the each cell's peripheral length. Statistical analysis demonstrates that Lf have positive correlation with cell roundness(Rn), area of cytoplasm(Ac) but a negative correlation with cellular area(Ar).

Bovine trypsin (EC 3.4.21.4) is a pancreatic serine protease that is widely used for biotechnological applications and pharmaceutical preparations due to its high specificity and good storage stability. Recombinant trypsin has been produced in a number of systems, including cell culture, bacteria and yeast. Since these expression systems have some problems (potential contamination with infectious agents and commercial-level production), recombinant trypsin molecules for the desired biotechnological applications have to be provided from new expression hosts. Recombinant bovine trypsin (rbtrypsin) was produced in transgenic rice cell suspension culture under the control of rice α-amylase 3D promoter (RAmy3D) which is highly expressed rbtrypsin (20 mg/L) during sugar starvation. The pure active rbtrypsin (5 mg/L) was purified by ion-exchange chromatography and it had a specific activity of 300 units/mg of protein in a standard TAME assay. These results means that the rbtrypsin form transgenic rice can replace animal-derived trypsin in the processing of pharmaceutical proteins. (This Study was supported by a grant from the Next Generation New Technology Development Project of the Ministry of Knowledge Economy).

Plant genetic engineering has led to production of plant-derived mAb (mAbP), which provides an inexpensive, efficient and safe alternative to traditional systems used for the production of recombinant antibodies. In this study, the expression levels of mAbP SO57 with or without ER-retention signal peptide (Lys-Asp-Glu-Leu; KDEL) in transgenic tobacco plants were analyzed. The expression levels of mAbP SO57 with KDEL were significantly higher than those in the without KDEL regardless of transcription level. Localization of mAbP SO57 with KDEL in the perinuclear region suggests that the mAbP with KDEL is localized in the ER. The mAbP without KDEL and mAbH had mainly Golgi type glycans, whereas the ER-localized mAbP with KDEL contained both oligomannose (47.6%) and Golgi type glycans (52.4%). The Fc domains of both purified mAbP (with and without KDEL) and human-derived mAb (mAbH) had similar binding activity to the FcγRI receptor (CD64). Both mAbP (with and without KDEL) had a shorter half-life than mAbH. However, both mAbP with and without KDEL were as effective at neutralizing activity of the rabies virus CVS-11 as the mAbH. These results suggest that the ER localization of a recombinant mAbP with a C-terminal KDEL sequence induces the reprogramming of glycosylation pathways and the enhancement of functional antiviral therapeutic antibody production in the plants.

Rock bream iridovirus (RBIV) have occurred frequently among cultured marine fish species in Southeast Asia (including S. Korea) and iridoviral disease has caused severe systemic disease with high mortality. The major capsid protein of RBIV is known to have a major effect on viral infection. In order to develop the edible vaccine against RBIV, MCP gene with KDEL endoplasmic reticulum retention signal was synthesized based on rice optimized codon usage (sMCP) by overlap PCR. The synthetic fragments, sMCP was inserted into a plant expression vector containing sugar starvation inducible promoter, RAmy3D, and introduced into the rice celli by bombardment-mediated transformation. The stable integration and transcriptional expression of the sMCP were confirmed by genomic DNA PCR, Northern blot analysis. Accumulation and production of major capsid protein in endoplasmic reticulum during sugar starvation in suspension-cultured cells was confirmed by Western blot analysis. These results suggest that transgenic suspension-cultured rice cells have the potential to be used as a edible vaccine against RBIV (This study was supported by Technology Development Program (108166-03-1-HD110) for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea).

Affinity tags have become indispensable tools for protein expression and purification, and tobacco etch virus (TEV) protease is widely used as a cleavage enzyme to produce recombinant proteins which are expressed as a fusion protein. Upon expression of TEV protease in Escherichia coli expression system, a recombinant protein was produced that exhibited proteolytic activity toward EGFP-hTNF-a (fusion protein expressed in E. coli) in vitro. In order to high-level expression in plant, we designed a mutant TEV protease(T17S,N68D, I77V, S219V) was fully codon-optimized based on Oryza sativa codon usage with mutations that resist autoproteolytic inactivation and improve the solubility. The resulting sTEV was subcloned into the plant expression vector and then introduced into rice callus via particle bombardment-mediated transformation. Fourteen transgenic lines were obtained and the transgene insertion was confirmed by genomic DNA PCR. sTEV protease mRNA and protein expression in transgenic rice suspension cells during sugar starvation under the control of RAmy3D promoter were confirmed by Northern and Western blot analyses (This study was supported by Technology Development Program (108166-03-1-HD110) of Ministry for Food, Agriculture, Forestry and Fisheries of the Republic of Korea).

Effects of environmental factors on cell growth and recombinant protein production have been widely investigated in Chinese hamster ovary (CHO) cell cultures. In addition, directed shifting of process parameters can be used as an optimization tool for providing higher cell and product yields. pH is a parameter known to influence cell growth, recombinant protein production, cell metabolism and protein glycosylation. In this work, we investigated the effect of pH on a recombinant CHO cell line expressing human albumin-erythropoietin (albumin- EPO), a highly glycosylated fusion protein. The culture pH was controlled at various levels (6.8, 7.0, and 7.2, respectively) by using 0.5 N NaOH. Maximum cell density reached up to 4.2 x 106cells/mL at day 4 by maintaining pH 7.2, which was 8% higher than that of control culture. The highest productivity was obtained at pH 6.8. However, lowering the culture pH led to cell aggregation. Based on these results, pH shift two-stage culture was performed. In the first half stage of cultivation, pH was controlled at 7.2 and the pH shifted at day 5 to 6.8 and 7.0, respectively. However, as reported in the previous studies, the culture pH at 37℃ did not influence albumin-EPO production. In conclusion, the pH shifting was found to be an efficient strategy for the increase of cell growth in CHO cell cultures. Therefore, it is expected that the simultaneous use of both pH and temperature shifting can improve the performance of CHO cells regarding cell growth, recombinant protein production and glycosylation.

Mucosal vaccination has been considered as an attractive immunization principle based on the consideration that mucosal surfaces are vulnerable for invasion by pathogen. In that sense, transgenic plant is noticed as promising carrier offering attractive option for production and delivery of mucosal vaccine. Classical swine fever (CSF) is a highly contagious viral disease of pigs and results in devastating financial losses. Because CSFV E2 glycoprotein mediates the viral attachment and penetration to susceptible cells, it is considered as a target for inducing protective immunity. In this study, in order to develop the vaccine materials for mucosal immunization against the CSFV infection, fully codon optimized E2 was introduced into the rice and expressed under the control of RAmy3D sugar starvation inducible promoter. The proteins expressed in the transgenic rice cultured cells effectively induced immune responses after intraperitoneal and oral administration. In addition, E2 conjugated with M cell targeting ligand, Co1, and we are currently analyzing immune responses induced by oral administration of E2/Co1-expressing rice calli (This study was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Agriculture, Forestry and Fisheries, Republic of Korea through the Agricultural R &D Promotion Center.)

In most countries, diabetes is the primary cause of end stage renal disease (ESRD). The glomerulus is the main filter of the nephron and is a major target of injury in diabetic nephropathy (DN). Although it is a major target of injury in DN, the mechanism of injury is inadequately understood. In this study, to understand glomerular injury mechanism leading to DN, the differences in STZ-induced rat glomerular proteomes in healthy, 6-weeks and 24-weeks old rats were analyzed via 2-dimensional electrophoresis (2-DE). To demonstrate stage of DN, body weight, blood glucose, and protein urea periodically were measured and pathological changes by Periodic acid-Schiff (PAS) staining were examined. To obtain glomerulus from kidney, magnetic beads were used and glomerular proteomes were analyzed by 2-DE and nano-LC-ESI-MS/MS. As a result, three proteins were down-regulated, one protein was up-regulated in STZ-induced group were compared with the control groups. These proteins will give base of mechanism for glomerular injury in DN.

Cryopreservation is a practical method of preserving plant cell cultures and their genetic integrity. Transgenic rice cells producing recombinant bovine trypsin were pretreated at an high osmotic condition (0.5M sucrose) to minimize formation and aggregation of ice during freezing. Transgenic rice cells were slowly prefreezing using Mr. Frosty freezing container in a deep freezer (-70℃) and preserved in liquid nitrogen (-196℃). For development of an optimal cryoprotectant for long-term storage, various concentrations of sucrose, glycerol and dimethyl sulfoxide (DMSO) to the precultured cells were investigated. The optimal condition of cryoprotectant was observed combination of 0.5M sucrose, 0.5M glycerol, 1M DMSO. The cell viability with this optimized condition was 62% after cell banking for 1day. The expression of recombinant trypsin in recovered callus from cryopreservation was also kept stable, and the production level was similar to that observed in noncryopreserved cultures. Acknowledgments This work was supported by a grant from the Next Generation New Technology Development program of the Ministry of Commerce, Industry and Energy, and by the National Research Foundation of Korea Grant funded by the Korean Government (2009-0070005)

The mAb CO17-1A recognizes the colorectal carcinoma-associated antigen GA733-2E, which is highly expressed on the surface membrane of human colorectal carcinoma cells. We have successfully developed a plant system for production of anti-colorectal cancer monoclonal antibody, mAb CO17-1A. The C-terminal KDEL (Lys-Asp-Glu-Leu) ER retention signal might be retained the recombinant glycoprotein carrying oligomannose glycans in the ER and enhances glycoprotein accumulation. In this study, we compared the expression levels of plant-derived mAb (mAbP) CO17-1A with and without KDEL in transgenic plants. We validated the expression of mAbP CO17-1A in transgenic plants via western blot analysis, and subcellular localization was monitored under confocal microscopy. Western blot analysis showed that the expression levels of the mAbP CO17-1A with KDELwere highly expressed than in the mAbP CO17-1A without KDEL. Furthermore, in ELISA with HCL116 colorectal carcinoma cells, the expression level of the mAbP with KDEL was significantly higher than that of the mAbP without KDEL. Flow cytometry (FACS) analysis showed that the Fc domains of both purified mAbP with KDEL and mammaldan-derived mAbM had similar binding activity to the FcγRI receptor (CD64). The Fc domains of the purified mAbP without KDEL had a lower binding activity to the FcγRI receptor (CD64) than the purified mAbP with KDEL. Glycosylation analysis revealed that mAbP CO17-1A without KDEL contained plant-specific N-glycans. On the other hand, mAbP CO17-1A with KDEL contained mainly oligomannose type glycans. Taken together, these results suggest that the ER retention signal peptiede (KDEL) can affects expression and glycan structures of mAbs.

In recombinant Chinese hamster ovary (rCHO) cell cultures, a particular attention has been paid to the design of an optimized medium in order to obtain high cell densities and enhanced productivity of recombinant proteins. For the achievement of these objectives, addition of various adding nutrient supplement was attempted and many results showed that the addition of various hydrolysates has been effective to enhance productivity. In this study, we added 3 g/L of yeast extract and phytopetone at day 3 to enhance the cell growth and production of albumin-erythropoietin(alb-EPO) supported maximum in rCHO cells. When we added the hydrolysates, it was found that yeast extract supported maximum cell density as well as alb-EPO concentration. Improvement of cell growth and alb-EPO production was 1.4-and 1.5-fold respectively. Lactate production, which is related with glucose consumption, was 2.4-fold lower than that of control culture without hydrolysates. These results suggested that the cultivation of rCHO cells with the addition of yeast extract was suitable for the improvement of cell growth and alb-EPO production.

Recently, disposable bioreactors have been increasingly incorporated into preclinical, clinical, and production-scale biotechnological facilities. However, there are few studies regarding their suitability for the production of pharmaceutical proteins in transgenic plant cell cultures. Plant cell culture have been regarded as a potential alternative production system for biopharmaceuticals. In this study, two kinds of disposable bioreactors were applied to transgenic rice cell cultures for the production of recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) and they were compared with 5-L stirred tank bioreactor system. The first one is a modified wave-mixed bag with a fritted sparger, and the second is an airlift disposable bioreactor. In both systems, fed-batch culture method was used for the efficient production of hCTLA4Ig by feeding concentrated amino acids. Dissolved oxygen level was regulated at 30% by supplementing pure oxygen with solenoid valve during the run. The results from wave-mixed and airlift disposable bioreactor showed similar maximum cell density (12.6 vs. 11.9 g DCW/L), doubling time (5.03 vs. 4.84 day) and maximum hCTLA4Ig concentration (44.5 vs. 43.7 mg/L). However, the time needed for obtaining maximum hCTLA4Ig level was much longer in airlift disposable bioreactor (26 vs. 34 days). With higher aeration rate (0.2 vvm), 1.7-fold higher specific productivity (0.355 mg/g DCW/day) was obtained. These results show that pnumatically driven disposable bioreactor system is better for the production of recombinant proteins from plant cell cultures.

The comet assay can theoretically be applied to every type of eukaryotic cell, including plant cells. Experiments were conducted to study the genotoxic effects of radiations on the genome integrity, particularly in Tradescantia [1]. Tradescantia tests are very useful tools for screening the mutagenic potential in the environment [2-3]. This study was designed to assess the genotoxicity of ionizing radiation using Tradescnatia-comet assay. Tradescnatia leaf samples were exposed to 0.1-50Gy. As an estimate of DNA migration, the length of the comet tail was recorded versus its head diameter under different conditions. In the non-irradiated sample the tails can be seen, but the DNA comets are short. The comet moment parameter which integrates both the morphometric parameter and the densitometric one may better reflect the amount of DNA which has been displaced from the nucleus. The comet assay has demonstrated its sensitivity as a technique for the evaluation of DNA damage among a variety of cell types, induced by a variety of physical and chemical agents [4]. The potential applications of the comet assay in such areas as genotoxicity, DNA repair, environmental biomonitoring and human monitoring are almost unlimited.

Various challenges have been attempted to increase the production of therapeutic proteins in mammalian cell culture. Process parameters such as pH, temperature and osmolarity have been shown to exert a significant effect on cell line performance. In this study, application of hyperosmotic pressure condition to the production of albuminerythropoietin (Alb-EPO) fusion protein by recombinant Chinese hamster ovary (rCHO) cells in suspension was investigated. Hyperosmotic pressure has been known to affect to the cell growth and the production of recombinant proteins. Generally, most cell lines are known to be damaged over 400 mOsm/kg. The osmotic pressure was shifted from control (320 mOsm/kg) to higher levels (380, 430, and 480 mOsm/kg) using 5 M NaCl at day 4 of cultivation. Cell growth and viability were found to be reduced with hyperosmolar condition. However, 380 mOsm/kg led to 1.2-fold increase in Alb-EPO production compared with that in control. In conclusion, hyperosmotic pressure was found to be a simple and effective parameter for increasing Alb-EPO expressed in rCHO cells.

Dandelion (Taraxacum platycarpum) has been widely utilized for medicinal purposes. However, the dandelion seeds are relatively difficult of germination under cultivation conditions, which hampers seedling propagation of dandelion plants and reduces the opportunity of usage of such a useful medicinal plant. Thus, in this study, in vitro conditions for the dandelion seed germination were optimized to enhance the germination rate. In seed washing steps, the sequential treatments with 20% of ethanol, 20% of NaOCl, and distilled water avoided microbial contamination with the highest in vitro germination rate (67.5%) from seeds sown in germination media. The media supplemented with 1.4 g/L of MS salts and 1% of sucrose significantly enhanced the germination rate compared to the media with 4.4 g/L of MS and 3% of sucrose. Sowing the seeds vertically in the optimized media supplement conditions, 1.4 g/L of MS salts and 1% of sucrose, gave the maximum in vitro germination rate (61%), which was almost three times higher than sowing seeds on a soil pot (23%). Our results indicate that the seed washing and sowing methods including germination medium supplements can be optimized to enhance in vitro seed germination of dandelion.

Recently, production of recombinant proteins has been studied using transgenic plant cell cultures. Transgenic rice cell cultures (Oryza sativa L.) were utilized to produce recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). General long-term preservation method for plant cells has been known to be the repeated subcultures. One drawback of this method is the genetic instability of transformed cell lines. For that reason, a cryopreservation method was developed for the long-term storage of the transgenic rice cells. DMSO has been known to be an effective cryoprotectant in freezing steps, but plant cells are damaged by cryoprotectants during thawing procedures. To solve this problem, an optimal method has been designed by using various cryoprotectant mixtures in freezing step and the cryoprotectants were removed by negative pressure in thawing steps. Optimization of cryoprotectant concentration was performed with range from 0 to 2 M. The best combination was found to be 1 M sucrose, 0.5 M glycerol and 0.5 M DMSO. The cell viability was 57.3% in that combination after 7days of cryopreservation. To remove toxic cryoprotectants in thawing steps, the times required for negative pressure treatment were maintained by 30 sec, 1 min, 2 min and 5 min, respectively. The best treatment time was found to be 2 min and cell viability was 84.7%. In conclusion, we could enhance the cell viability in cryopreservation of transgenic rice cells by optimizing cryoprotectants-related steps.

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced inducibly by sugar starvation using RAmy3D promoter in transgenic rice cell suspension cultures. Although RAmy3D promoter is known to be the strongest promoter in plant systems, main obstacle is that the target protein was produced in sugar-free conditions. To improve the production of hCTLA4Ig, we applied anoxia conditions with 30 mM of glucose to the production phases. The anoxia conditions with 30 mM of glucose at the production phases markedly improved cell viability under anoxia conditions. A viability level of more than 65% could be maintained for up to 30 days. Furthermore, the repression of the RAmy3D promoter by residual sugar in the production of hCTLA4Ig was not observed under anoxia conditions with 30 mM of glucose. In addition, the production periods of hCTLA4Ig was expanded up to 30 days, and the maximum production levels of hCTLA4Ig were 29 mg/L under anoxia conditions and 14 mg/L in aerobic conditions, respectively. Under anoxia conditions with 30 mM of glucose, alcohol dehydrogenase II gene was found to be strongly expressed. Therefore, we suggest that anoxia conditions could be used to achieve energy source by ethanolic fermentation for the improvement of the productivity of hCTLA4Ig in transgenic rice suspension cell cultures.

Plant cells have several advantages as a host for the production of therapeutic proteins, such as low production cost and safety from animal pathogens. Recently, many therapeutic proteins are produced by using transgenic plant cell cultures. Transgenic rice cell cultures (Oryza sativa L.) with inducible RAmy3D promoter have been used to express recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), a soluble fusion protein used for the treatment of rheumatoid arthritis. Induction of the RAmy3D promoter is strongly performed by sugar starvation and the transgenic rice cells secrete hCTLA4Ig into the medium. Sodium butyrate (NaBu) has been used to enhance the production of foreign proteins in Chinese hamster ovary (CHO) cell cultures due to its harmlessness, easy manipulation and cost-effectiveness. However, it may also inhibit cell growth and induce cellular apoptosis. In this study, for the enhanced production of hCTLA4Ig, effects of NaBu on cell growth and protein production were investigated. Feeding concentration (0, 0.5, 1, 5 mM) of NaBu as well as the supplementation of additional sodium pyruvate (87 mM) as an energy source were optimized and studied at induction phase. In conclusion, the production of hCTLA4Ig could be significantly enhanced by adding 1 mM NaBu with additional energy source in transgenic rice cell cultures.

With effective cancer treatment or immunotherapy by target specificity and high affinity, therapeutic antibodies have been hot issues. Unlike other recombinant proteins ; erythropoietin (EPO), interferons and growth factors, dosage range of therapeutic antibodies is needed above 5 mg/kg. In addition, productivity of recombinant Chinese hamster ovary (rCHO) cells compared with microorganism is so low, thus experiments to improve productivity is essential. rCHO cells expressing chimeric antibody against CD20 antigen were amplified and selected with methotrexate (MTX) in adherent cultures ,and rCHO cells later adapted to serum-free, protein-free and chemically defined media. First of all, media selection experiment is carried out. After medium with high productivity is chosen, the effects of sodium butyrate (NaBu) and temperature conditions were studied in an attempt to increase antibody production from suspension adapted rCHO cells. The NaBu, despite its cytotoxic effect, increased the production of recombinant antibody (rAb) in rCHO cell culture. When the culture temperature was shifted from 37 ℃ to 30 ℃ at a culture time of 72 h, final rAb concentration was about 6 times higher than production at 37 ℃. Establishing a culture condition of high concentration of rAb production, mass production is attempted by Pneumatic bioreactor system (PBS) performed 2 L and 13 L scale. These scale up culture processes were successfully performed. Based on the results, foundation of development for culture process of rCHO cells producing recombinant antibody was established.

A rice α-amylase 3D system, which is induced sucrose starvation, has been previously reported to generate a good yield of recombinant protein in transgenic rice cell suspension culture. However, this induction system was high produced not only foreign protein but also undesirable proteins, rice α-amylase and cysteine proteinase (CysP), into the culture medium. A rice α-amylase is a dominant protein, with 43% of total secreted proteins and CysP was a major secreted protease in rice cell suspension cultures following induction via sugar depletion. Increasing the yield of recombinant proteins, protecting the produced proteins from protease, and diminishing expensive protein purification steps are important research goals in plant biotechnology. In the present study, rice amylase and CysP genes were eliminated almost completely via RNA interference (RNAi) technology to develop rice cells as a more efficient foreign protein expression system. The effects of RNA interference were characterized at both SDS-PAGE and Western blot analysis against CysP antibody and quantitative real-time RT-PCR analysis. Dependent upon the amylase and CysP ihpRNAi line, amylase and CysP decreased by approximately 90% at both the proteins and mRNA levels. However, the phenotype of ihpRNAi lines was not distinguishable from wild-type rice plants under in vitro and ex vitro growth conditions.

Insect cell-baculovirus vector system is one of the promising method for the production of recombinant proteins. Baculovirus produces heterologous recombinant proteins under polyhedrin promoter and is safe because it has a limited host range. Insect cells are easy to cultivate and have post-translational modifications. The development of Porcine Cholera Marker Vaccine is urgently required, because economical losses are very big on pig breeding market in Korea as well as around world. So insect cell-baculovirus vector system was used in this study for production of Porcine Cholera Marker Vaccine. Since Serum has been commonly used in insect cells culture, 10% TNM-FH medium was used for the production of recombinant Porcine Cholera Marker Vaccine by using several insect cell lines of Sf9, Sf21, and Hi-Five. The Sf9 cell growth was highest when insect cells were inoculated at 1x106 cells/mL and 5x105 cells/mL. The effects of multiplicity of infection and harvest time on marker vaccine production were investigated in each case of individual cell lines. As a result, the highest productivity of Porcine Cholera Marker Vaccine achieved was 22.5μg/mL in the condition of 0.001 MOI and 5 days of harvest time with Sf9 cells. However, the highest virus titer obtained was 7.25x106 pfu/mL at the condition of 10 MOI and 3 days of harvest time with SF9 cells.

Transgenic plant cell cultures are an attractive expression system for the production of industrial and pharmaceutical proteins because of their advantages in safety and low production cost. Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced and secreted when sugar was depleted in culture medium by transgenic plant cell lines (Oryza sativa L.) using RAmy3D promoter. Formation of reactive oxygen species (ROS) is an inevitable consequence of the aerobic organisms such as animal and plant cells. ROS gives rise to biotic and abiotic stresses. Therefore, ROS levels need to be controlled and the addition of antioxidants can reduce apoptosis caused by ROS which generated during cell cultures with aeration. Many researchers reported the effects of antioxidants, such as glutathion, ascorbic acid (vitamin C), and vitamin E on plant cells. In this study, we investigated the effects of N-acetylcystein, ascorbic acid and reduced glutathion on cell growth and protein production in transgenic rice cell cultures under oxidative stress. Effects of antioxidants with various concentrations were studied by comparing protein production levels using ROS markers. In conclusion, long-term survival of transgenic rice cells and with enhanced hCTLA4Ig production were possible by reducing the level of ROS with the additions of various antioxidants in transgenic rice cell cultures.

The autodisplay technology is one of the surface display methods of proteins or peptides to the E.coli outer membrane. In the autodisplay techonolgy, proteins or peptides are expressed as a recombinant protein in the autotransporter domain of a protein called AIDA-I (adhesin involved in diffuse adherence) from E.coli. In this work, the autodisplay of streptavidin, a protein with biotin-binding activity, is presented. The autodisplay of streptavidin was confirmed by SDS-PAGE of the outer membrane proteins, and the number of autodisplayed streptavidin molecules on a single E.coli cell was estimated by using densitometric analysis. The biotin-binding activity of the autodisplayed streptavidin was estimated after treating fluorescence labeled biotins by using fluorescence microscope and FACS. The biotin-binding activity of the E.coli with autodisplayed streptavidins was compared with similar sized magnetic beads with covalently immobilized streptavidins. Finally, the outer membrane with autodisplayed streptavidin was isolated and layered on a 96-well microplate and the biotin-binding activity was estimated to determine whether it is applicapable to immunoassays.

A new covalent immobilization method for small proteins and short peptides is presented by using parylene-H film which, is a polymer of p-xylene having formyl groups. The covalent coupling of proteins to the parylene-H film is produced by only one step of incubation of proteins or peptides without additional coupling reagents. In this work, the parylene-H film is coated on a 96-well microplate for immunoassays. The immobilization efficiency to the parylene-H film was compared with the conventional physical adsorption by using human chorionic gonadotrophin protein and a small peptide called circulated citrullinated peptide as model molecules. Additionally, the applicability of this immobilization method for short peptides is demonstrated by detecting autoantibodies in rheumatoid arthritis patient serum.

Neurotransmitters has been implicated in a number of physiological and behavioral functions, such as control and regulation of affective behavior, sleep, vigilance, reward system, motor and cognitive function, and neuroendocrine function. The nanotechnology-based bio-barcode- amplification analysis offers an innovative approach to detect dopamine. We evaluated the efficacy of this protein-detection method in detecting dopamine in normal and oxidative stress damaged dopaminergic cells. In this study, a coupling of DNA barcode and bead-based immnunoassay for detecting neurotransmitter with PCR-like sensitivity is reported. The method relies on magnetic nanoparticles with antibodies and nanoparticles that are encoded with DNA and antibodies that can sandwich the target protein captured by the nanoparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated to remove the conjugated barcode DNA. The DNA barcodes were identified by PCR analysis. The concentration of dopamine in dopaminergic cell can be easily and rapidly detected using bio-barcode assay. The bio-barcode assay is a rapid and high-throughput screening tool to detect of neurotransmitter such as dopamine. Acknowledgement: The research was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (2009-0080860) and by the Nano/Bio Science &Technology Program (M10536090001-05N3609-00110) of the Ministry of Science and Technology (MOST) and by Ministry of Environment of the Republic of Korea as "The Eco-technopia 21 project".