Earticle

Protein drugs have been widely used in various human diseases which were cancer, diabet, immune etc. However protein drugs have some problem to use therapeutic drugs (short half life, high cost). Thus several delivery technologies have been reported, microsphere, nanoparticle, ionic complex etc. Among the technologies, Poly ionic complex (PIC) has been used in protein drug delivery via charge-charge interaction. The advantage of PIC is easily prepared, but disadvantage of PIC is easily decomposition in human body condition(high concentration of salt). Several different types of PIC has been introduced to overcome this problem, which was conjugated-hydrophobic(1) and crosslinkmoiety(2). In this study, we developed novel PIC system that was conjugated thermosensitive moiety in biodegradable charged polysaccharide. And the property of thermos-ensitive-PIC was confirmed by UV-spectrophotometer, Zetasizer, and fluorescence. Thermo-sensitive PIC is successfully overcome the problem of earlier PIC system. Thus this system would be a very useful strategy for a therapeutic protein delivery.

The effects of water soluble tacrolimus-PEG conjugates (KI-102) on the insulin-dependent diabetes mellitus and systemic lupus erythematosus were investigated. The stable ranges of KI-102 were at pH 4.0-4.5 and 4℃. Area under the concentration time curve, the time of maximum concentration, and the maximum concentration were was 43.4ng.hr/mL, 0.85 hr and 8.1 ng/mL, respectively. These results were similar to those of FK506. The plasma glucose concentration in the 4.32mg/kg of KI-102-fed group at 170 days of age was decreased to 7.5 mmol/L, which was similar to that of 0.6mg/kg of FK506. There were no incidences when 86.4 mg/kg of KI-102 was administered after 24 weeks. About 60% of the β- hydroxybutyrate concentration at 43.20mg/kg-fed group, 24% of the triglyceride concentration at 43.20mg/kg-fed group, and 30% of the cholesterol concentration at 43.20mg/kg-fed group were decreased, respectively. The immunosuppressive effects of systemtic lupus erythematosus were depended on KI-102 concentration. At 180.0 mg/kg of KI-102-fed group, the serum anti-dsDNA antibody activity was about 64% of decreased compared to control. The urinary albumin concentration at 180.0 of KI-102 mg/kg-fed group was about 81% of decrease compared to that of control. These results indicate that KI-102 may be practically applicable as prodrug of FK506.

Protein nanoparticle is one of effective vehicles for drug delivery because of its biocompatibility and biodegradable property. Among various proteins, human serum albumin (HSA) is widely used as building block of protein nanoparticles because it is abundant, stable, nonantigenic, and available for covalent modification due to its functional groups. HSA is introduced to cells mainly by receptor-mediated endocytosis, however, HSA nanoparticles have limited application to cells only having albumin receptor. In this study, recombinant 30Kc19, cell penetrating protein originated from silkworm was used to overcome this limitation. 30Kc19-HSA nanoparticles were formed by dropwise addition of ethanol to aqueous protein solution with continuous stirring and then were crosslinked using glutaraldehyde. 30Kc19-HSA nanoparticles had uniform spherical shape and stability in phosphate buffered saline and cell culture media. 30Kc19-HSA nanoparticles had negligible toxicity to animal cells. Successful uptake of 30Kc19-HSA nanoparticles to HeLa and human fibroblast cells was observed using fluorescence microscopy. In addition, transfection vector loaded 30Kc19-HSA protein nanoparticles had successfully delivered pDsRED2 gene to cells. It is expected that 30Kc19-HSA protein nanoparticles could be used as versatile tool for drug delivery to various cells.

UV responsive liposomes were prepared with egg phosphatidylcholine (egg PC) and 7-acetoxycoumarin. 7-acetoxycoumairn (7-ATC) show an photo reactivity under UV irradiation. Under the light wavelength at 365nm, 7-ATC formed dimer and under the light of wavelength at 254nm, 7-ATC cleaved to monomer again. Dimerization was investigated from liposome with 7-ATC and without 7-ATC. The optimum ratio of 7-ATC to egg PC was 1:8. 5(6)-carboxy fluorescence (CF) was released from liposome under 254nm UV irradiation. Due to the fluorescence dye was not stable under 365nm UV irradiation. 7-ATC dimer was prepared under 365nm UV irridation first, used to prepared. Release of CF containing in liposome with 7-ATC dimer was higher than that in liposome without 7-ATC dimer. 7-ATC dimer was located in liposome bilayer membrane because it was hydrophobic. When irradiate at 254nm UV light, 7-ATC dimer become monomer and liposome bilayer membrane was very unstable. As a result, liposomes were disintegrated so that CF was released.

Thiazolidinedione analogue, CT-8, has been reported to be a potent inhibitor of human 15-hydroxyprostaglandin dehydrogenase (15-PGDH). In continuing attempts to develop highly potent 15-PGDH inhibitors, a series of thiazolidinedione analogues with different substituents on phenyl rings were synthesized and tested. Of the compounds produced, those in the low nanomolar range were most effective. In particular, introduction of halogen atoms at the phenyl ring of CT-8 greatly improved its inhibitory efficacy. For structure-activity analysis, the effects of adding cyclohexyl with different chain lengths to the hydroxyl groups of 5-(3-substituted-4-hydroxybenzylidene)thiazolidine-2,4-dione were tested. Removal of the methylene group between the cyclohexyl ring and oxygen of 5-(3-substituted-4-(2-cyclohexylethoxy)benzylidene)thiazolidine-2, 4-dione decreased its inhibitory potency. It appears that two methylene groups between the cyclohexyl ring and oxygen of 5-(3-substituted- 4-hydroxybenzylidene)thiazolidine-2,4-dione are optimal for inhibitory activity. The most potent inhibitor of this series of compounds is compound 5, (5-(3-chloro-4-(2-cyclohexylethoxy)benzyl)thiazolidine-2,4-dione), with an IC50 of 0.008 μM.

Human kringle domains (KDs) are ubiquitous domains known as binding modulator for various biomolecules with seven flexible loops, and very recently it was successfully demonstrated that KDs could be engineered toward target-specific binding proteins as an alternative protein scaffold. Here, we report the development of efficient expression system of KD derivative (KD548) as a promising anti-cancer agent in Escherichia coli host and fed-batch cultivation for its preparative scale production. Even though KD548 needs three disulfide bonds for correct folding, expression in cytoplasm allowed the highly soluble and much enhanced production than periplasm. For the efficient expression, four sets of expression systems consisting of different promoters (lac or T7) and different fusion tag (10xHis or FLAG) were examined. Among them, use of T7 promoter-FLAG tag fusion system resulted in much higher production than others in shake flask cultivation as well as in fed-batch cultivation performed in 6.6 L jar bioreactor. When cells were induced at higher cell density (OD600=100) and complex feeding solutions were supplemented, cells density and production yield could be improved significantly which were also much higher than that by previous Pichia host (~ 8 mg/L).

Recombinant human growth hormone (hGH) has been used as a therapeutic agent to treat children's growth disorders and adult growth hormone deficiency. hGH has been known to be highly susceptible to rapid proteolysis and has a short half-life of only a few hours in human blood stream. Here we developed an experimental and rational method to select modification sites in hGH polypeptide, where any changes (glycosylation or site-directed mutagenesis) can be applied. First, we selected several putative permissive sites and performed site-specific glycosylations for improved proteolytic stability. hGHs with site-specific mutations were expressed in Saccharomyces cerevisiae, and their glycosylations were confirmed by SDS-PAGE analysis and glycosidase treatment. The activities of glycosylated hGHs were tested using Nb2 cell proliferation bioassay. When compared with the wild-type unglycosylated hGH, the glycosylated hGH did not lose its biological activity. The proteolytic stability of glycosylated hGH was also tested by western blot analysis after treatment of plasmin. As a result, glycosylated hGH showed enhanced stability compared with the wild-type hGH. These results indicate that glycosylation at internal permissive sites was successful to develop protease-resistant hGH.

Quantitative measurement of anti-HBs is used to evaluate the response to hepatitis B vaccination in health care workers and also used to potency test for several immunoglobulin preparations as an effectiveness factor. Comparison of 5 assay methods for anti-HBs is performed to improve potency assay of Human Immunoglobulin G(HuIgG) preparation. This study established EIA method for anti-HBs comparing with current MEIA method. Three commercial EIA kits were compared with co-laboratory studies including the HuIgG manufacturers and Korea FDA using national standard (NS) and international standard (IS) of anti-HBs. Resulted similar reproducibility (average CV=5%, recovery >96%) of each kits however, Enzygnost was selected as an optimal EIA kit which showed the most stable precision (CV=4.1%) when the negative plasma was applied as diluent for sample and standard. Even though negative plasma and 5% human serum albumin (HSA) showed compatible reproducibility, negative plasma gave better precision (CV=4.1% vs 7.1%) and lower background. Using the optimal EIA kit, validation were performed including intra- and inter-day assays, inter-laboratory assay and inter-method comparison with 22 representative HuIgG preparations. The samples consist of 9 lot of maltose added HuIgG, 3 lot of HuIgG, 5 lot of Anti-HBV HuIgG and 5 NS spiked samples (total 22 samples). As a result intra day precision was 4.6±2.0% and inter day precision was 7.7±5.3 for EIA (N=180 for 5 days), 3.7±1.9 for AxSym (N=10 for 5 days) and 1.6±0.8 for IMx(N=6 for 2 days). As an accurate test with the results of NS spiked samples, the mean recovery was 100.1±12.6%. The statistical analysis showed that the detection limit of EIA was below 10 IU/L and the range was 10~150 IU/L. The linearity of dose response curve with NS were confirmed (R2>0.998) and robustness was proved based on the spiking assay with representative samples (100.1%) which showed no matrix effect. Therefore, the EIA assay was validated and showed relevance to MEIA including two automated instruments; IMx and AxSYM of Abbott. As a conclusion the EIA assay could replace the current MEIA method.

Exenatide is a glucagon-like peptide-1 receptor agonist, a small peptide secreted in salivary gland of Gila monster. A marketed synthetic version of exenatide is Byetta® approved in 2005 for the treatment of type 2 diabetes. Since Byetta® is twice a day drug administered by injection, long-acting formulation of exenatide is needed for the improvement of patients compliance. In this study, we designed long-acting exenatide/ NexPTM protein, where exenatide was fused to NexPTM in order to extend the half-life in plasma. Recombinant exenatide/NexPTM was expressed in CHO-K1 cell at a high level and the expressed protein was confirmed by SDS-PAGE as well as by immunoblotting with monoclonal antibody specific to exenatide. Pharmacokinetic study in Sprague-Dawley rats showed that the fusion to NexPTM increased the half-life of exenatide twenty times longer compared to exenatide without fusion. Exenatide/NexPTM effectively suppressed the blood glucose level in db/db mouse and DIO(diet-induced obesity) mice. These results indicate that exenatide/NexPTM could be effectively used as a long acting drug for type 2 diabetes therapy.

Fucoidan which is contained in various species of brown seaweed such as sea mustard, kombu, ecklonia cava, and etc. appears to inhibit the production of igE. That is the source of the reduced secretion of allergic mediators, for example, asthma, hay fever, allergies, dermatitis. Fucoidan is a very useful material to relieve the symptoms of allergic diseases and prevention. In this study, it is confirmed that anti-atopic lotion containing fucoidan cures the damaged hairless mice skin caused by atopic dermatitis through observing the outward appearance of the hair less mice skin and measuring the thickness of the skin induced by atopic dermatitis, which was gradually reduced, using a digimatic indicator. Through the evaluation of immunological, fucoidan suppressed the IgE and IL-4 expression, but promoted IFN-γ expression so that make a balance between Th cell that turns out to be constituted. In addition, the weight of the spleen divided by body weight, then according to T lymphocyte, IgE and IL-4 expression inhibited the ability, and IFN-γ expression were identified.

In this study, 18 natural products were screened via anti-angiogenesis experiments, and 1 candidate product was identified, Corni fructus, which exerted dose-dependent inhibitory effects against angiogenesis, adipogenesis, and cell adhesion. C. fructus extract (CFE) exhibits an angiogenesis inhibitory effect superior to that of the EGCG from green tea leaves. The expression level of angiogenesis and adipogenesis-related signal molecules in the western blotting was reduced by increasing the amount of added CFE. Moreover, a diet supplemented with CFE was deemed more effective in inducing weight loss in LB mice than a representative synthetic diet drug, orlistat, which incidently caused the side effect of denuding the mice of their hair. These results indicate that C. fructus may prove to be a useful anti-adipogenic compound, and these in vitro results may be reflected later under in vivo conditions.

This study reports on a drug-eluting stent (DES) that has an anti-proliferation and an anti-coagulation in biodegradable polymer and nature polymer hydrogel-coated onto a cobalt-chromium stent. The DES was prepared by ultra-sonic spray coating the bare metal stent with PLGA loaded sirolimus and Alginate hydrogel loaded heparin top-coat to treat against restenosis and coagulation, respectively. The double-layered drug coated stent was characterized in vitro for surface morphology and coated thickness by scanning electron microscopy (SEM) and confocal microscopy. Additionally, the in vitro degradation and agents release study of the different cross-linking degree alginate hydrogel as CaCl2 concentration were examined. The results show DES can controlled release both anti-proliferation drug ( sirolimus ) and anti-coagulation agent ( heparin ). This result expect reduction restenosis because sirolimus control release. Sirolimus control release was reported of reduction restenosis as smooth muscle cell proliferation suppression.(1) Also, heparin control release was expect anti-activation of platelets and anti-coagulation. Heparin release or immobilization was reported of anti-thrombosis effect as improved blood compatibility. (2) Therefore, this study Alginate top-coated sirolimus-eluting stent will be expected provide effective coronary artery disease treatment. Acknowledgment : This study was supported by a grant of the Korea Healthcare technology R&D Project Ministry for Health, Welfare &Family Affairs, Republic of Korea. (A085012).

Alginate has been used as popular biomaterials in the forms of hydergel, microporous beads and fibers for biomedical applications. However, more versatile applications were limited by its fragility in spite of excellent biocompatability. Fiber reinforced composites have often been suggested to improve mechanical characteristics and alginate hydrogel was reported to be successfully reinforced by chitin whisker [1] and alginate fibers [2] to improve their physical properties.In this report, a concentrated calcium salt solubilizing silk fibroin was used for ionotropic curing alginate and for preparing fibroin fibers in the hydrogels. Confocal laser scanning microscope analysis indicated that the FITC stained alginate beads prepared with fibroin solution contained both short fibroin fibers and a network-like fibroin fibers of similar diameters. Methanol treatment inducing β-sheet formation increased the mechanical strength of the fibroin reinforced alginate hydrogels. A versatile method of forming fibroin-fiber networks is under development.

Silk fibroin is an insoluble structural protein from Bombyx mori. It can be regenerated as a fibroin solution to form a variety of biomaterials, such as gels, sponges and films, for medical applications [1]. A submicron-scale fibroin microsphere with core-shell structure has been developed [2]. Cellular uptake of submicron-scale particles has been reported for polystyrene microspheres [3] and for gold nanoparticles [4]. In this report, submicron scale microspheres were prepared and its cellular uptake by 3T3 cells in vitro was investigated. Confocal laser scanning microscopy and tomographic analysis revealed that fibroinmicrospheres were successfully incorporated into the 3T3 cells in vitro and they were localized in cytoplasm. The efficiency and rates of fibroin-microspheres were investigated using fluorescence-activated cell sorting.

In this study, 18 natural products were screened via anti-angiogenesis experiments, and 1 candidate product was identified, Siegesbeckiae herba, which exerted dose-dependent inhibitory effects against angiogenesis, adipogenesis, and cell adhesion. S. herba extract (SHE) exhibits an angiogenesis inhibitory effect superior to that of the EGCG from green tea leaves. The expression level of angiogenesis and adipogenesis-related signal molecules in the western blotting was reduced by increasing the amount of added SHE. Moreover, a diet supplemented with SHE was deemed more effective in inducing weight loss in LB mice than a representative synthetic diet drug, orlistat, which incidently caused the side effect of denuding the mice of their hair. These results indicate that S. herba may prove to be a useful anti-adipogenic compound, and these in vitro results may be reflected later under in vivo conditions.

The solvent evaporation process is a convenient method for controlling the morphologies of paclitaxel. Amorphous paclitaxel was simply made by dissolving paclitaxel in methylene chloride/methanol (98/2, v/v) and in relatively non-polar solvents (t-butyl methyl ether, pentane, acetonitrile/hexane (1/2, v/v), methylene chloride, chloroform, toluene). On the other hand, crystalline paclitaxel was made by dissolving paclitaxel in a special polar solvent containing a small amount of water. However, when we used only methanol, we got mixed morphologies of paclitaxel made of both the crystalline and amorphous forms. Their physicochemical properties were investigated by X-ray powder diffraction (XRPD), scanning electron microscopy (SEM), and high performance liquid chromatography (HPLC). The initial water content of amorphous paclitaxel and crystalline paclitaxel was determined for 0.65 wt% and 5.85 wt%, respectively. The hygroscopic property of crystalline paclitaxel was very changeable in all given humidity (15, 60, 95 RH%) during storage. Dissolution profiles for paclitaxel showed that amorphous paclitaxel measured the highest solubility in water and its solubility held most stable during the measurements. The residual solvent could be reduced to the maximum allowed value (600 ppm for methylene chloride, 3,000 ppm for methanol) of guidance for the International Conference on Harmonization (ICH) by spray drying.

Mono-PEGylated VM501, human interleukin-11 mutein, was developed to provide prolonged in vivo blood circulation. VM501 is an advanced human Interleukin-11, of which efficacy and safety was confirmed already in phase I and II clinical trials in China. VM501 is currently preparing phase III for the treatment of chemotherapy induced thrombocytopenia. However, like most interleukins, VM501 has a short half-life in the body. Therefore, daily dose is needed to produce platelets effectively. To provide longer circulation time in vivo, a polyethylene glycol chain was attached via amine specific conjugation method. Hence, VM501 has three lysine residues and one amino-terminus, there are four possible conjugation sites. To purify mono-PEGylated VM501, improved resolution of purification method is needed. Several commercially available resins were tested for differentiation of mono-PEGylated VM501 over di-PEGylated and/or non-PEGylated VM501. Let alone dynamic binding capacity needed to be maintained or improved also. Here we introduce some results among tested resins. One of the resin provided better resolution over di-PEGylated version and the non-PEGylated VM501 was well separated. The overall yield was increase more than 3 times than the conventional regular resin.

In this study, a microwave-assisted extraction (MAE) method was used to recover the anticancer agent paclitaxel from plant cell cultures, and the extraction efficiency of the paclitaxel was determined using various organic solvents (acetone, chloroform, ethanol, methanol, and methylene chloride) and solvent concentrations. Methanol provided the highest recovery of paclitaxel (~93%) and resulted in the most severe rupturing of the biomass surface during MAE. Most of the paclitaxel (>99%) was recovered using a methanol concentration of 90% (water content: 10%), suggesting that the addition of a small amount of water improves the efficiency of MAE. Furthermore, analysis of the surface of the biomass using an electron microscope revealed that the higher the recovery of paclitaxel, the more severe the damage to the biomass surface. A comparison of the extraction efficiency between MAE and conventional solvent extraction (CSE) showed that with CSE, only up to 54% of the paclitaxel could be recovered in one extraction whereas with MAE, most of the paclitaxel (>99%) in the biomass could be recovered in one extraction.

Cellulase enzymes have attracted considerable attention in recent years due to their great biotechnological and industrial potential. Cellulase enzymes provide a key opportunity for biomass utilization through the bioconversion of the most abundant cellulosic material into the simplest carbohydrate monomer. Nowadays, the sources of cellulase-producing bacteria have been broadened into the presence of symbiotic bacteria in herbivorous animal and also from marine. Takifugu rubripes or known as Puffer fish is a unique poisonous vertebrate but nevertheless is considered a delicacy in Korea. This dietary habit considers Puffer fish as host of cellulase-producing bacteria, especially on its gut. In the present study an attempt has been made to search for the cellulolytic bacteria in the gut of Takifugu rubripes. Fifty five microorganisms have been isolated using 1% (w/v) Carboxymethyl cellulose (CMC) as substrate. Congo red dye test and DNS method were then used for screening the extracellular cellulase activity of the strains. Among them, TD6 strain has shown the highest performance in term of cellulase activity. In order to evaluate the optimum culture condition of the isolate TD6 for cellulase production, the strain was grown at various temperatures, pH, carbon sources, and nitrogen sources. Under optimum condition, the maximum specific activity of 0.90 U/mg protein was achieved after growth the strain with 1.5% CMC at 45oC pH 6 for 3 days, respectively. Based on 16S rRNA analysis it is proposed that the strain was Bacillus subtilis TD6.

Hydroxy fatty acids can be used as starting materials of resin, waxes, nylons, plastics, lubricants, biopolymer, biodiesel, and additives in coatings and paintings. The recombinant E. coli was used to convert oleic acid to 10-hydroxy stearic acid in order to produce hydroxy fatty acids. The converted 10-hydroxy stearic acids were isolated and purified through biochemical process. The recombinant E. coli was cultivated in Luria-Bertani medium at 37℃, 200rpm. When the OD600nm of the culture reached 0.6, 0.1 mM of IPTG was added. The culture was incubated in the condition of shaking at 150rpm, 16℃ for 12hrs. Tris-HCl buffer was used for the conversion of oleic acid into 10-hydroxy stearic acid at 30℃, pH 7.5. The converted 10-hydroxy stearic acids were tested to find the optimal extraction solvent, solvent ratio and extraction times. 10-Hydroxy stearic acids were extracted better when used ethyl acetate, chloroform, ethyl ether, and hexane. The efficiency of recovery is higher when extraction process is proceeded twice or third times. Also, it is more efficient when the ratio between culture medium and solvent is same.

Supercritical carbon dioxide (SC-CO2) is very effective in removing oils from a variety of seed matrices. In this work sunflower seed was used as the substrate for the extraction of oil by supercritical CO2. Any scale up procedure for supercritical fluid extraction process needs the optimization of the process variables. The process variables were optimized by Response Surface Methodology. Four variables, such as temperature, pressure, particle size and solvent flow rate were optimized by 24 full factorial central composite design and subsequent analysis and model validation by a second-order regression equation. The response was taken as the amount of oil obtained. Extracted oil was determined as a function of the extraction temperature, pressure, solvent flow rate and particle size.

Superparamagnetic iron oxide particles functionalized with iminodiacetic acid (IDA-SPION) with varying diameters ranging from 350 to 450 nm were synthesized by covalently grafting GLYMO-IDA on silica coated iron oxide superparamagnetic nanoparticles (silica coated SPION). The size and/or ligand density of IDA-SPION is adjustable by changing the parameters (i.e. sonication time to disperse magnetite growth seed or TEOS addition rate) during the sol-gel based synthesis process. Cu2+-charged IDA-SPION (Cu2+-IDA-SPION) exhibits size and metal ion ligand density dependent binding affinity and capacity for a model protein, bovine serum albumin (BSA). As the size of Cu2+-IDA-SPION increases, higher maximum BSA adsorption capacity (Qm) and dissociation constant (Kd) are exhibited, indicating that the increase in BSA adsorption capacity is coupled with compromised affinity towards the target protein.1 This research provides an insight to synthesizing IDA-SPIONs with controllable size and protein adsorption property, thereby providing a simple method capable of addressing the various requirements in a wide range of protein purification or impurity removal applications.

Gloshedobin is one of the promising therapeutic thrombin like enzymes, potentially useful for the treatment of blood clotting disorders through their anti-coagulant function. However, it is difficult to obtain a sufficient amount of the enzymes from the nature, so we use Escherichia coli to offer a route for the rapid and economical production of recombinant proteins. However, over-expression of recombinant gloshedobin in E. coli led to intracellular accumulation as solid aggregates known as inclusion bodies (IBs). We would like to develop novel refolding cocktail based refolding strategies by harnessing in vitro realization of a m olecular chaperone network for renaturation of solubilized but denatured gloshedobin in a manner to mimic cellular folding machinery. In order to redesign the traditional refolding schemes with the use of refolding cocktail containing various molecular chaperones (e.g. DnaK, DnaJ, GrpE and ClpB), two types of chromatographic column are prepared; 1) Packed bed column (PBC), and 2) Monolithic column (MC). Column-based refolding efficiencies and kinetics are assessed at various compositions and recirculation flowrates of the refolding cocktails and their performances at several protein concentrations compared to those of the traditional ones. Overall the MC based novel refolding strategy harnessing unpurified molecular chaperone cocktails proves superior to the traditional refolding schemes in multiple aspects including productivity, process economics and process intensification.

Potent antimicrobial substances having immuno-modulatory activity were isolated from a Korean soil actinomycetes. These compounds were purified by reverse-phase HPLC with 60% acetonitrile linear gradient. Obtained three compounds showed antimicrobial activity especially against gram positive bacteria including VRSA. MICs of the compounds against 16 species were determined using the agar dilution method. Antimicrobial activity of these compounds were stable in pharmaceutical processing conditions e.g. against heat (up to 90 ℃/ 4 hr, 121℃ /15 pound and 15min), freezing (-20℃) and pH treatment (pH 3-9). Broth agar dilution plate containing CS392s were pre-incubated at 37°C for 0–2 h before inoculation with control strains. These compounds which have strong anti-VRSA (and VRE) showed immuno-modulatory activities. They dose dependently inhibited NO production and inducible NO synthase (iNOS) expression in RAW 264.7 macrophages activated with lipopolysaccharide (LPS). To investigate the signaling pathway for NO inhibition by these compounds, we examined nuclear factor-κB (NF-κB) translocation in RAW 264.7 cells. We concluded that these compounds inhibit the synthesis of pro-inflammatory cytokines and suppress LPS-induced NF-κB activation in RAW 264.7 cells.

Succinic acid is one of the useful platform chemical materials that can be converted into various valuable materials. The bio production process of succinic acid is a promising method to reduce environmental problems. However the separation processes to purify organic acid from fermentation broth is one of the most energy consuming process. The development of cheap and high yields separation process is crucial to consist whole bio-production process of succinic acid.Herein, we utilized electro dialysis(ED) and water splitting electro dialysis(WSED) with ion-transfermembrane to separate succinic acid from fermentation broth. Produced succinic acid from Mannheimia succiniciproducens is interfered metabolism cycle which is designed to produce succinic acid. Therefore recover of succinic acid with proper rate which is compensate the producing rate is needed to maintain its productivity. We demonstrated constant voltage operation condition with various initial ammonium succinate concentrations and voltages both ED and WSED method. We can concentrate ammonium succinate solution up to 80g/L using 20g/L fermentation broth solution using ED operation with 18V condition. The following WSED operation of concentrated ammonium succinate can transform into pure succinic acid without further acidification process and interference of other organic acids such as pyruvic acid or lactic acid. In this study we can purify succinic acid from fermentation broth properly using environmental friendly operations which can reduce the usage of chemical materials.

Fossil fuels as the source of transport fuels are now widely recognized as unsustainable due to problems with global warming and limited availability. Renewable biofuels including biodiesel, bio-ethanol, bio-hydrogen, bio-oil and bio-gas have been focused on as an alternative energy source. A variety of biomasses from different sources have been investigated as the feedstock for the production of different biofuels. Microalgae have a higher photosynthetic efficiency than other biomasses. In addition, microalgae have been promoted as a future source of transportation fuels primarily because of their potential to produce up to 10 times the amounts of oil per acre compared with vegetable oils, animal fats and crops. In this study, supercritical fluid extraction of microalgae was performed to study the influence of process parameters such as extraction time, temperature, pressure and ultrasonic power on the yield and composition of the extracted oil. In addition, the effect of the size, amount and packing method of glass beads on the oil extraction was investigated in detail.

Lignin is one of the plant biomass to protect trees against chemical and biological attack. Therefore, lignin is increasing interest in the improvement of quality of life that lead to eco-friendly technology demands. Since organic compounds of nano-materials were reported to be antibacterial activity, we extracted the lignin particles of nano-particles under 100 nm of average diameter. To prepare the PLA film with a high antibacterial activity, we attempted to fix lignin particles on PLA film by a coating process. Lignin particles were extracted by an organic solution method from wood materials by using 96% dioxane as a solvent. This extracted lignin particle was blended with the solution including GMA and then coated on PLA film with a bar coater. Thickness of coated layer was about 4~5 um and the total thickness including PLA film was controlled as 40~50 um. Antibacterial activity of the PLA film with lignin particles was enhanced and its hardness was about 2~3 H. Lignin is one of the plant biomass to protect trees against chemical and biological attack. Therefore, lignin is increasing interest in the improvement of quality of life that lead to eco-friendly technology demands. Since organic compounds of nano-materials were reported to be antibacterial activity, we extracted the lignin particles of nano-particles under 100 nm of average diameter. To prepare the PLA film with a high antibacterial activity, we attempted to fix lignin particles on PLA film by a coating process. Lignin particles were extracted by an organic solution method from wood materials by using 96% dioxane as a solvent. This extracted lignin particle was blended with the solution including GMA and then coated on PLA film with a bar coater. Thickness of coated layer was about 4~5 um and the total thickness including PLA film was controlled as 40~50 um. Antibacterial activity of the PLA film with lignin particles was enhanced and its hardness was about 2~3 H.

High level expression of recombinant protein often leads to the accumulation of inactive and improperly folded proteins as insoluble aggregates in the host cell, so-called inclusion bodies (IBs). Thus, an additional protein refolding process is required to convert IBs to water-soluble active protein. Ionic liquids (ILs) are organic salts composed of anion and cation. In particular, ILs which do not crystallize at room temperature are called room temperature ionic liquids (RTILs). Tunable physiochemical properties of RTILs such as hydrophobicity and polarity leads the expansion of their applications in various chemical and biological processes.1 Recently Summers and Flowers applied ethylammonium nitrate (EAN) on dilutionbased refolding of lysozyme and achieved 90% of refolding yield by suppressing the aggregation of denatured protein.2 In this study, the effect of hydrophobicity and substituted anions of ILs as refolding additives on lysozyme refolding was investigated. Furthermore, lysozyme refolding process was optimized in terms of the refolding time, concentration of RTILs and operating temperature. Refolding yield was proportionally decreased with alkyl chain length of RTILs. MS-based RTILs was more effective than BF4-based RTILs. The refolding yields of lysozyme were improved over 100 % and 81 % when 0.5 M and 1.0 M of MS- and BF4-based RTIL was added in refolding buffer, respectively. The optimum refolding temperature was found at 25℃.

Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor (TGF)-β superfamily, can uniquely induce ectopic bone formation. BMP-2 like other bone morphogenetic proteins, plays an important role in the development of bone and cartilage. It’ involved also in cardiac cell differentiation and epithelial to mesenchymal transition. Recombinant human BMP-2 (rhBMP-2) on an absorbable collagen sponge has been shown to be a safe and effective replacement for iliac crest bone graft when used with a threaded fusion device in anterior lumbar interbody arthrodesis. Insect cell protein expression system is considered to have several advantages in high capacity, flexibility, and similar human glycosylation pattern. For these reasons, the insect cell culture with baculovirus transfection is a useful system for expression of mammalian recombinant protein. hBMP-2 was expressed in insect cell baculovirus system. The cDNA fragment encoding hBMP-2 was amplified for insertion into baculovirus expression vector, pFastBacTMHT. The hBMP-2 expression cassette was inserted into a parent bacmid in DH10Bac (E. coli). The isolation recombinant bacmid was transfected to Sf9 insect cell. The expression of hBMP-2 from the transfected cell was confirmed with immunoblot analysis. Furthermore, we will validate the insect cell expression system as an efficient production reactor for hBMP-2.

IgY (Immunoglobulin Yolk) is a specific antibody in egg yolk, and it protects human body from virus and antigen. There are a lot of egg yolk components such as lipoprotein and protein. To separate IgY, HPLC (High Performance Liquid Chromatography) and precipitation were used in a batch mode and SMB (Simulated Moving Bed) was adopted for continuous purification of yolk proteins. IgY and other proteins in yolk were separated by using three-zone SMB chromatography. Before performing SMB experiments, batch chromatography and PIM (pulse input method) were performed to find operation parameters and adsorption isotherms. The results of batch chromatography were compared with simulated results using Aspen chromatography. To find the most suitable separation condition in SMB chromatography, simulations in m2-m3 plane on the triangle theory were carried out. m2 = 0.18, m3 = 1.0 and △t = 419 s are the best conditions for the highest purity of IgY. With this operating parameters (flow rate in three zone and switching time), the purity of raffinate results in 98.39 % from Aspen chromatography simulation. Most of the simulation reached steady-state with in second recycle.