Earticle

Enterokinase is a heterodimeric serine protease consisting of 82~140kDa heavy chain and 35-62kDa light chain. The light chain is a catalytic subunit of enterokinase, which consists of a chymotrypsin-like serine protease. Because of high specificity for the amino-acid sequence (Asp4)Lys, enterokinase has a great potential as a fusion protein cleavage reagent. Recombinant HEKL was produced in transgenic rice cell suspension culture with the rice amylase 3D promoter. RAmy3D promoter is highly expressed by sugar starvation. The expression of recombinant HEKL as mRNA and protein in transgenic rice cell was detected by Northern and Western blot analyses, recombinant HEKL was observed about 43 kDa in Western blot analysis. The activity of recombinant HEKL was determined by cleavage of the fusion protein EGFP-hTNFa as substrate, which contained a enterokinase cleavage site (DDDDK) between two moieties. Under optimal conditions the hTNFa product with expected size of 17kDa was well detected on Western blot analysis. These results demonstrate the use of HEKL derived from plant cell suspension culture as proteases (This Study was supported by a grant from the Ministry of Knowledge Economy).

Recently, the production of recombinant proteins (plant-made pharmaceuticals) has been studied using transgenic plant cell cultures. The transgenic rice cell lines (Oryza saitva L.) were utilized to produce recombinant human Cytotoxic T-Lymphocyte Antigen 4-Immunoglobulin (hCTLA4Ig) in this study. General long-term preservation method for plant cells is known to be the repeated subcultures. One drawback of this traditional method is genetic instability of transformed cell lines. Cryopreservation method was developed for the long-term storage of the transgenic plant cells that could maintain the genetic stability. However. additional step is essential in plant cells damaged by ice crystal formation because of high water content up to 90%. For the purpose of solving this problem, various osmotic agents (sucrose, mannitol, and sorbitol) were applied to reduce the water content of plant cells. Molar concentration of each agent was adjusted at 0.2 M, 0.4 M, and 0.6 M, respectively. F/D ratio was decreased and cell viability was rapidly decreased at 0.6 M due to high osmotic stress. When 0.4 M of osmotic agents were added, F/D ratio was 6.06, 7.92, and 7.06 respectively at day 2, which was 0.50-fold, 0.34-fold, and 0.42-fold decrease compared to that of control. Relative cell viability was the best in the case of 0.4 M sucrose treatment. Therefore, we can develope the optimal pretreatment method for transgenic plant cell cryopreservation.

Murine inositol-requiring enzyme 1 alpha (IRE1α) appears to function as a proximal sensor of the endoplasmic reticulum (ER) stress. To test if IRE1α has any functional role during the dieldrin-induced ER response and subsequent apoptosis, first, the wild type IRE1α cDNA and a mutant IRE1α lacking the c-terminal kinase domain (ΔIRE1α) were isolated by RT-PCR cloning, which is the routine cDNA cloning procedure. After cloning into the expression vector (pFLAG-CMV), their protective or apoptotic effects were tested by transient expression analysis under normal and dieldrin-treatment conditions. Dopaminergic neuronal cells expressing ΔIRE1α showed lower expression of BiP/GRP78, which is thought to be an ER chaperone relieving the ER stress. However, the cells expressing wild-type IRE1α significantly elevated the BiP/GRP78 expression on account of dieldrin treatment. In addition, ΔIRE1α expressing cells showed higher rate of morphological features of apoptosis than cells expressing wild-type IRE1α or mock (empty plasmid) after treatment with dieldrin. These results suggest that c-terminal kinase domain of IRE1α is a key component in the battle against ER stress and neuronal cell death.

Chinese hamster ovary (CHO) cells have been used as a popular mammalian host for the commercial production of therapeutically important proteins. The effects of increased productivity of secreted proteins expressed in CHO cells following treatment with histone deacetylase inhibitor (HDACi) was studied.1) Sodium butyrate, sodium propionate and valproic acid are effective inhibitors of histone deacetylation.2) These compounds induce histone hyperacetylation and enhance the expressed mRNA transcription level of target protein. In addition, HDACi can inhibit proliferation of the cells and induce apoptotic cell death and arrest at the G1 phase of the cell cycle.3) To determine the effect of HDACi on the production of recombinant albumin-erythropoietin (alb-EPO) and cell cycle, various compounds were added to the cultures of exponentially growing cells. Among the inhibitors tested, sodium propionate was found to be the most efficient. We optimized the concentration of sodium propionate to enhance the production of alb-EPO with minimum growth inhibition. It was observed that 1 mM sodium propionate was optimum considering cell growth as well as alb-EPO production. Sodium propionate arrested the cell cycle in G1 phase. Compared with the culture without sodium propionate, the culture with 1 mM sodium propionate resulted in a 1.2-fold increase in alb-EPO concentration on day 6. These results suggest that the application of sodium propionate for the commercial production of recombinant proteins in CHO cells is feasible.

Factor X, a vitamin K-dependent plasma protease occurs by specific interaction with components of the blood coagulation pathway. Factor X is activated in vitro by a factor X activator isolated from Russell's viper venom (RVV). Factor Xa cleaves after the arginine residue in its preferred cleavage site Ile-(Glu or Asp)-Gly-Arg, widely used to remove fusion taqs from expressed proteins. In this study, 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 of the Ministry of Knowledge Economy).

Bovine trypsin is a pancreatic serine protease with substrate specificity based upon positively charged lysine and arginine side chains. Trypsin is widely used for commercial purposes to digest or process other proteins. Plant production system of recombinant proteins offers a way to replace animal-derived proteins with a safe and economical alternative. 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 during sugar starvation. The expression level of rbtrypsin was 20mg/L. It was purified from transgenic rice cell suspension culture medium by hydrophobic interaction chromatography and size exclusion chromatography. From 1L flask culture, 0.55mg pure active rbtrypsin was obtained. The enzyme activity of purified rbtrypsin was generally characterized by zymogram. These results demonstrate a possibility to use the rbtrypsin derived from rice cell suspension culture for commercial purposes. (This Study was supported by a grant from the Next Generation New Technology Development Project of the Ministry of Knowledge Economy).

In order to examine the effect of Dandelion (Taraxacum coreanum) extracts on the liver cancer induced by N-nitrosodiethylamine (DEN) in Rats. The animals were divided into three groups. The animals in the normal (Nor) group were fed basal diet. control (Con) group were administered with DEN (200 mg/kg, i.p.). Dandelion extracts (DDA, DDB) group treated with dandelion extract (30, 60 mg/kg/day) for 8 weeks after DEN. Enzymic antioxidants, such as superoxide dismutase (SOD) and catalase levels were determined in all the groups of animals. The activities of SOD were significantly increased in the Con, but the activities of catalase were decreased in the Con, but the activities of SOD and catalase were increased in the IJH. In the immunohistochemistry observation, treatment of Dandelion extract reduced the rates of p53 immunoreactivity. These results suggest that administration of Dandelion extract suppress or retard DEN-induced liver cancer.

Papaver somniferum produces a variety of benzylisoquinoline alkaloids (BIAs) and has been known as the major plant source producing morphinan alkaloids including thebaine, codeine and morphine. Since the earliest stage of in vitro cultures of P. somniferum, the production of morphinan alkaloids in tissue culture has been attempted. Undifferentiated callus cultures of P. somniferum have been reported to lose their ability to synthesize morphine, but the reasons has not been clearly understood. Somatic embryogenesis from undifferentiated callus of Papaver somniferum contributed to differential expression of gene transcripts and proteins involved in the benzylisoquinoline biosynthesis accompanying in vitro morphine production. After initiation of differentiation of callus, overall gene transcripts on the BIA biosynthetic pathway of .somniferumaccumulateinsomaticembryos; and 37.5 μg of morphine from 1 gram of dried somatic embryos was measured using LC tandem mass spectrometer. Among genes on the morphinan alkaloids biosynthetic pathway, salutaridine reductase (SR) which is stereo-specific cytosolic enzyme reducing salutaridine into salutaridinol before thebaine, doesn’t accumulate in undifferentiated callus at all; whereas both SR gene transcript and protein are detected in somatic embryos. Major latex protein, the most abundant protein in plants’ laticifer where morphinan alkaloids accumulate, was expressed during somatic embryogenesis and could be adopted as an index of differentiation of callus according to somatic embryogenesis. This study represents that differentiation during indirect somatic embryogenesis of P.somniferum activates morphinan alkaloids biosynthesis, which is confirmed by comparative profiling of metabolites, gene transcripts and proteins involved in BIA biosynthesis.

Sepsis is the severe cause of death that is characterized by wide-spread inflammatory response in whole body. Previous studies have suggested that peroxisome proliferator-activated receptor γ (PPARγ) ligands may inhibit the synthesis of pro-inflammatory cytokines. Therefore, this study investigated whether natural PPARγ ligand, 15-deoxy-delta 12, 14-prostaglandin J2 (15d-PGJ2), and the FDA-approved synthetic PPARγ ligand, rosiglitazone, may also modulate the inflammatory response and improve mortality in sepsis rat model induced by cecal ligation and puncture. Sepsis induced by cecal ligation and puncture resulted in approximately 25% mortality in rats within 4 days. However, preadministration of 15d-PGJ2 (1 mg/kg) or rosiglitazone (5 mg/kg) significantly improved survival rate and over 80% animals were alive at 4 days. Although the serum levels of cytokines, such as IL-6, IL-10 and TNF-α were increased after cecal ligation and puncture, pre-treatment with 15d-PGJ2 or rosiglitazone significantly suppressed those cytokines production. These results demonstrated that the PPARγ ligands have beneficial effects altering inflammatory processes and can protect from sepsis.

Sodium salicylate, a main metabolite of aspirin, has been shown to protect neurons. Animals injected with 6-hydroxydopamine (6-OHDA), a hydroxylated analogue of dopamine, are well-accepted model revealing nigral degeneration and have served as an animal model of Parkinson’s disease. In this study, 6-OHDA model was used to test the neuroprotective effects of sodium salicylate in relation to suppression of microglial activation. 6-OHDA induced microglial activation and a significant extent of microglial activation remained at least up to six weeks following a single injection of 6-OHDA into the medial forebrain bundle. However, the response to 6-OHDA was significantly attenuated by sodium salicylate administration. In addition, the survival rate of substantia nigra dopaminergic neurons was better when sodium salicylate treatment was initiated after 6-OHDA lesion than when sodium salicylate was administered prior to the 6-OHDA lesion. These findings suggest that sodium salicylate can act as a negative regulator of the inflammatory response in dopaminergic system.

Recently, as a result of drastic progress in nanotechnologies, the use of nanoparticles and quantum dots has significantly surged to various applications from scientific- to industrial field. In particular, the nanoparticles has expanded to be used in our daily life such as garment, tooth brush, and cosmetics. However, toxicity by nanoparicles has been controversially argued and, to date, great efforts have been devoted to prove the toxicity of nanoparticles. Titanium dioxide (TiO2) nanoparitcles have physical properties diffusing and reflecting UV radiation; due to these properties, cosmetics of sun-cream contain TiO2 nanoparticles to block UV radiation. In this work, the toxicity of silicagel-coated TiO2 nanoparicles on skin- and lung cell was investigated using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) assay- and confocal microscopy for live & dead of cells. The skin- and lung-cell were parcelled out from Korean Cell Line Bank. In addition, E. coli growth curve of TiO2-treated cells was measured. We observed that TiO2 nanoparticles impedes the cell growth (using MTS), and dead cells increase depending on the concentration of TiO2 nanoparticles using confocal microscopy. It was also confirmed that E. coli growth curve is influenced by the concentration of TiO2 nanoparticles. Based on the results mentioned above, we discuss the toxicity and the safety of TiO2 nanoparicles for the human cells.

The objective of this study was to identify the proteins involved in myogenesis and adipogenesis. Global proteins were monitored in skeletal muscle and adipose tissue between bull and steer, respectively. Through proteomic analysis and comparison, we identified 4 differentially expressed (two-fold or more) proteins from skeletal muscle in steer, down-regulated A, B, C and up-regulated D, and also identified two up-regulated proteins from adipose tissue, E and F. In vitro, after myogenic differentiation of a bovine cell line, the mRNA expression of A, B and D did not changed, but that of C increased significantly (P<0.05). After adipogenic differentiation of a bovine cell line, the mRNA expression of F did not significantly vary, but the expression of E showed about 1.7-fold increase. Therefore, we suggest that C could be indicative of positive regulator of myogenesis, and E indicative of positive regulator of adipogenesis since they showed a differential expression before and after myogenic and adipogenic differentiation.

Primary culture of hepatocytes is an in vitro model widely used to investigate various aspects of liver physiology and pathlogy. Type I rat tail collagen is usually used as a coated material for hepatocyte culture. It promotes attachment and growth of hepatocytes. Here, we performed the potential use of a recombinant mussel adhesive protein (MAP), conjugated with type I collagen derived mimetic peptide as a cell proliferation and differentiation in hepatocyte. Analyses of cell morphology by light and scanning electron microscopy demonstrate that a culture system, in which key components of the extracellular matrix (ECM) have been optimized, can support a differentiated phenotype of primary rat hepatocytes. Furthermore, the results indicate that the MAPTrixTM-C (Collagen I mimetic) is similar morphology compare with other ECM systems. Using MAPTrixTM-C (Collagen I mimetic), differentiated cell morphology can be maintained for at least two weeks. Tight clusters of spherical cells with a lumen appear within the first day of culture in the MAPTrixTM-C (Collagen I mimetic). Cells will not spread, but remain in clusters throughout the entire culture period of two weeks. Cellular functions typical of differentiated hepatocytes are maintained for a at least two weeks as shown by continued expression of cytochrome P450, a hemoprotein involved in the oxidative metabolism of various compounds, an important function of the liver. Therefore, the MAPTrixTM-C (Collagen I mimetic) is suitable for use as a cell-adhesion material in hepatocyte culture.

Adhesion of cells on surface is a basic and important concept in the fields of neural cell culture and morphogenesis of the nervous system. An appropriate material for cell adhesion generally leads to improved differentiation of neuronal cells. Here, we investigated the potential use of a recombinant mussel adhesive protein (MAP), conjugated with laminin derived mimetic peptide as a cell-adhesion material, cost-effective and high-level production in Escherichia coli. Extracellular matrix (ECM) affect process outgrowth, branching of neurites, and profile of neural cells. MAPTrixTM ECM-laminin coatings support cell with the similar diverse morphology compare with other ECMs. ECMs affect adhesion, MAPTrixTM ECM-laminin, InvitrogenTM human fibronectin and GibcoTM mouse laminin coatings have greater adhesion compare with poly-l-lysine. ECMs affect expression and growth of axons and dendrites. Greatest number of dendrites and axons are seen on MAPTrixTM ECM-laminin, InvitrogenTM human fibronectin and GibcoTM mouse laminin coatings. ECMs affect polymerization of actin filaments. High levels of F-actin are produced on MAPTrixTM ECM-laminin, InvitrogenTM human fibronectin and GibcoTM mouse laminin coatings. ECMs affect expression of the glutamate neurotransmitter receptor. The highest levels of GluR1 are found on MAPTrixTM ECM-laminin, InvitrogenTM human fibronectin and GibcoTM mouse laminin. Therefore, the MAPTrixTM ECM-laminin is suitable for use as a cell-adhesion material in neural cell culture.

A rice cell suspension culture system with the RAmy3D promoter, which is induced by sucrose starvation, has been previously utilized to produce large quantities of recombinant proteins. However, using rice as production system for therapeutic proteins requires modification of their N-glycosylation pattern because of the immunogenicity of plant-specific sugar residues. In this study, for generating glyco-engineered rice as production host for therapeutic glycoproteins, an intron-containing selfcomplementary hairpin RNA-mediated post transcriptional gene silencing was used to obtain a targeted down-regulation of the endogenous glycosyltransferase genes in rice cell suspension cultures. N-linked glycans from the generated RNAi lines were identified and their structures were compared with those isolated from the wild type suspension cells. The Δ3FT/XT glyco-engineered line with significantly reduced xylosylated and/or core fucosylated glycan structures. (This Study was supported by Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries and also by a grant from the Ministry of Knowledge Economy)

Quality control of glycan profiles is a critical issue in the biogical and pharmaceutical industry. Recently, the glycan profile method which combined with Girard's reagent T (GT) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was exploited for qualitative and quantitative analysis of glycans. In this study, we present a rapid and high-throughput N-glycan release, purification, methyl esterification and GT derivatization method using 96-well plate based procedures. The N-glycans were released from model glycoprotein using a polyvinylidene fluoride membrane filter plate, than subsequently loaded onto porous graphitic carbon containing 96-well plate to remove the salts. The purified N-glycans were neutralazated by methyl esterification using 96-well plate, than GT derivatizated for generate a permanent cationic charge to the reducing end of neutral glycan. finally, the N-glycans were characterized by qualitative and quantitative analysis using matrixassisted laser desorption/ionization time-of-flight mass spectrometry. It is believed that the established 96-well plate platform described here may contribute to the rapid screening of potential N-glycan biomarkers.

Cordyceps sinensis is one of the most valued herbs in traditional chinese medicine. The anti-hyperglycemic activiity of the exopolysaccharides (EPS) produced from submerged mycelial culture of Cordyceps sinensis (Cs) was studied in a type II diabetic animal model (C57BL/6J ob/ob). This study was designed to determine whether EPS improves clinical signs of type II diabetes in ob/ob mice. After treatment of ob/ob mice with Cs-EPS at doses of 200 mg/kg body weight, the fasting blood glucose levels decreased by 47% after 63 days. The results of oral glucose tolerance test (OGTT) showed that the glucose levels returned approximately to baseline after 120 min in EPS-treated mice. Furthermore, food intake, body weight, total plasma cholesterol and triglyceride concentrations in ob/ob mice treated with EPS were significantly decreased. Plasma insulin concentrations were significantly increased by EPS treatment. In addition, we investigated the expression pattern of leptin with important effects in regulating body weight, metabolism and reproductive function. It was reveled that Leptin mRNA was expressed in adipose tissue from EPS-treated ob/ob mice. Immunoblot analysis revealed that the EPS increased the expression of leptin in adipose tissue and plasma. These results indicated that use of these compounds should be considered as a dietary supplement to control hyperglycemia in patients with type II diabetes.

The development of Porcine Cholera Marker Vaccine is keenly required, because economical losses were very big on pig breeding market in the world. The baculovirus expression vector system was used for the production of heterologous recombinant proteins as a powerful expression vector system. Undefined fetal bovine serum has several defects following downstream processing, contamination and variation in commonly using insect cell cultures. Therefore, the marker vaccine is also required to be produced by serum free media. It is possible for commercially produced several SFM (Sf 900 II, Sf900 III, Ex-Cell 405, Ex-Cell 420, Ex-Cell Titer High, Express- Five, SFX-insect, Serum Free and Protein Free insect medium-1) to achieve high cell density of Sf9 cells and high yield of recombinant baculovirus. The production of porcine cholera marker vaccine was investigated by infection of recombinant baculovirus into Sf9 cells in various SFM. An optimum SFM for the production of marker vaccine were searched. The result of the study showed that two SFM (sf900 III and ex-cell 420) yielded similar productivity to 10% serum supplemented TNM-FH medium. Since Sf900 III showed slightly higher yield than Ex-Cell 420, SF-900 III was selected for optimum serum free medium for the production of porcine cholera marker vaccine resulting recombinant baculovirus titer of 4.25x107 pfu/cells at 0.1MOI.

Recently, the rice α-amylase 3D promoter system activated under sucrosestarved conditions has emerged as an alternative system for the production of recombinant proteins. However, the influence of N-linked glycan structures related to sugar starvation conditions has not yet been investigated. The N-glycan patterns obtained from glycoproteins grown in growth medium with sucrose (+S) were different from those obtained under sucrose starvation (-S). The mass spectra of +S glycans exhibited eleven N-glycan patterns: MMXF3, GnMX, GnMXF3, GnGnX, AMXF3, GnGnXF3, AGnXF3, (FA)GnXF3, AAXF3, (FA)(FA)X, and (FA)(FA)XF3. The five N-glycans that disappeared under -S conditions were GnMX, GnGnX, AAXF3, (FA)(FA)X, and (FA)(FA)XF3. The N-glycan pattern of hGM-CSF derived from rice suspension cell culture was similar to that of -S condition, and all N-glycans contained plant-specific β1,2-xyolse, core α1,3-fucose, and some Lewis a epitopes. High-mannose type N-glycans were not detected under either condition (-S or +S) among the secreted glycoproteins. (This Study was supported by Technology Development Program of MIFAFF, and also by a grant from the Ministry of Knowledge Economy)

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 (shMCP). The resulting fragments, shMCP was inserted into a plant expression vector containing sugar starvation inducible promoter, RAmy3D, and introduced into the rice calli by Agrobacterium mediated transformation. The stable integration and transcriptional expression of the shMCP were confirmed by genomic DNA PCR, Northern blot analysis and RT-PCR. Accumulation 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) of MIFAFF).

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 work was supported by the grant from MIFAFF)

To enhance therapeutic protein production in Chinese hamster ovary (CHO) cells, Sodium butyrate (NaBu), one of the various histone deacetylase inhibitors, is widely used as a culture media additive. However, it is known that it induces cell growth inhibition and apoptosis in a dose-dependent manner. Of late, a significance of autophagy has increased in the field of mammalian cell culture because autophagy is known to be related to the programmed cell death mechanism. In this study, we determine the effect of NaBu on apoptosis and autophagy and investigate the role of autophagy under NaBu treatment in recombinant CHO cells producing erythropoietin (EPO). NaBu induced a drop in the viability and cell growth, and increased apoptotic characteristics in a dose - and time - dependent manner. Concurrently, a high concentration of NaBu also induced autophagy independently with nutrient deprivation. To elucidate the potential role of autophagy induced by NaBu, a representative autophagy inducer and inhibitor were treated with NaBu simultaneously, which showed that autophagy had the role of a positive cell survival mechanism under NaBu treatment. This positive function of autophagy might be mediated by the autophagic removal of damaged mitochondria based on the results of the recruitment of a mitophagy protein, Parkin, to the mitochondria. Taken together, autophagy was observed in rCHO cell culture under NaBu treatments with apoptosis as a pro-survival mechanism, which emphasized the necessity for engineering of pro-survival aspects of autophagic pathway to overcome the NaBu-induced apoptotic cell death.

Affinity tags have become indispensable tools for protein expression and purification, and tobacco etch virus (TEV) protease emerge as a useful reagent with wide application in the cleavage of recombinant fusion proteins. 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, TEV protease was fully codon-optimized based on Oryza sativa codon usage with mutations that resist autoproteolytic inactivation and improve the solubility. The resulting shTEV 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. shTEV protease mRNA and protein expression in transgenic rice suspension cells during sugar starvation under the control of RAmy3D promoter were confirmed by Northern and SDS-PAGE analyses (This Study was supported by Technology Development Program for Agriculture and Forestry of MIFAFF).

In the last two decades, ordered nanostructures on the solid surface have generated considerable interests owing to their unique electronic, optical, and biological characteristics. In the present work, Zinc selenide (ZnSe) nanocrystals were synthesized in the cavity of the apoferritin from horse spleen (HsAFr), and the two-dimensional ZnSe-ferritin nanodots were prepared on modified silicon surface. The arrays on the silicon surface provided the PL emission peak for ZnSe quantum dots in ferritin core. In addition, some neurotransmitters can be conjugated to the arrays and the PL spectrum changes accompanying by the conjugation reaction, the fluorescence intensity of ZnSe quantum dots in ferritin core was investigated. It was found that the intensity was dependent on the concentration of the neurotransmitter. When the array was conjugated with GABA, a significant enhancement of the fluorescence occurred and the enhancement factor was a linear function of the GABA concentration despite the presence of glutamic acid. In reality, it was found that the two-dimensional ZnSe-ferritin nanodot array could be a potential sensor media for GABA detection. This approach is superior to the old method to measure neurotransmitters in vivo which is complicated by the low concentration of neurotransmitters and suffers from overlapping resonance signals by other substances.

L-Homocysteine (L-Hcy) is a thiol containing amino acid that is formed as an intermediate when methionine is converted to cysteine. L-Hcy is known as an important independent risk factor of cardiovascular disease. The early diagnosis and treatment of hyperhomocysteinemia is critical for public health care and metabolic disease patients including homocystinuria. It has been used for the determination of total L-Hcy concentration by several methods, such as HPLC, stable isotope dilution, and enzyme immunoassay. However, they are all time-consuming and require highly skilled technical staffs. Here we developed new L-Hcy assay method by using a hybridoma producing monoclonal antibodies that specifically recognize L-Hcy. Monoclonal antibodies specific to L-Hcy were produced using L-Hcy coupled to BSA with glutaraldehyde as an antigen. The enlarged lymph nodes from rats injected via hind footpads with an emulsion of antigen and Freund’s adjuvant were used for cell fusion. Hybridoma clones secreting the monoclonal antibodies were selected by ELISA.

Plasma L-homocysteine (L-Hcy) is known to be a risk factor of various diseases. The measurement of plasma L-Hcy concentration is important for the prediction of several clinical conditions including homocystinuria, atherosclerosis, and thrombophilia. Accordingly, the interest in Hcy determinations has increased in research laboratories and companies. Several companies are already producing the kits for the detection of L-Hcy concentration. However, those kits need enzymatic conversion process of Hcy to S-adenosyl-L-homocysteine. We have generated monoclonal rat anti-Hcy antibody that specifically recognize the Hcy, so it will give big merits to simplify the detection method by elimination of reduction and conversion process. We could assay the localized endogenous L-Hcy by immunofluorescence in calf pulmonary artery endothelial (CPAE) cell for the first time by the direct monitoring of Hcy in living cells using our developed antibody. And competitive ELISA data suggest that the sensitivity for the detection of free L-Hcy by developed anti-Hcy monoclonal antibody is less than 100 nM level which is about ten times higher than previous detection kit.

DNA microarray technology enables a significant expansion of the molecular identification and characterization of multiple species in food safety, medical, agricultural and public health. In this study, we focused on development of species identification DNA microarray chips for pathogens. Three food-borne pathogens were selected for a random genomic DNA fragment-based chip, Staphylococcus aureus, Salmonella enterica and Bacillus cereus. At first we followed probe development protocols, the strategy of a random genomic DNA fragment, and constructed the proto-type DNA microarrays which have about 50 probes of each strain. It was validated and optimized for the detection of significant strains. To discriminate significant probes in the proto-type DNA microarray, we have successfully tested seven food-borne pathogens such as, Escherichia coli O157, Enterobacter sakazakii, Listeria monocytogenes, Yersinia enterocolitica, Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, and Alicyclobacillus cycloheptanicus including E. coli as a non-pathogen. Finally, we have completed the probe contents after removing the probes cross-talked with other non-specific species, and successfully developed a specific DNA microarray which can be used to detect 3 specific target pathogens and discriminate non-specific pathogens.

Thin film which consisted of metalloprotein via various chemical linker was fabricated to investigate linker-length effect between immobilized protein and Au surface. A metalloprotein was utilized the myoglobin which has redox property due to the iron ions of heme group in this research. The surface thickness was confirmed by surface plasmon resonance spectroscopy, and the morphology of surface was investigated with scanning tunneling microscopy. The redox property of immobilized myoglobin via chemical linkers having with lengths was carried out by cyclic voltammetry. The redox peaks were shifted due to the incremental length of chemical linkers because electron transfer between myoglobin and gold surface was interrupted by these materials as a capacitor. In these results, it could be applied to regulate redox property of biomolecule for realizing bioelectronic devices. Acknowledgement: This research was supported by The Nano/Bio Science&Technology Program(M10536090001-05N3609-00110) of the Ministry of Education, Science and Technology (MEST), by the Original Technology Research Program for Brain Science through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (2009-0093907), by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (2010-0000845).

Increased obesity characterized by excessive accumulation of adipose tissue is associated with insulin resistance and type 2 diabetes. Vaspin is an adipokine that has been isolated from visceral adipose tissue recently identified as a function of insulin-sensitizing effects. Previous studies demonstrated that its concentrations are related with insulin resistance. It means that vaspin is a useful marker for the diagnosis of type 2 diabetes. Several analytical formats for adipokines using antibody such as ELISA and western blotting have been reported because there is a high demand of an early diagnosis for diabetes and disease caused by obesity. In this study, aptamer-based SPR biosensor, which has been known to be advantageous over other antibody-based assays, was developed to detect the vaspin levels. For this purpose, the ssDNA aptamer candidates that contain conserved sequence in random region were selected from initial random ssDNA library pool containing 3x1014 molecules through FluMag SELEX process. It is confirmed that one of the aptamers screened shows high specificity and affinity (Kd = 0.303 ± 0.028 μM) to vaspin based on the measurement of the RU (response unit) using SPR. The minimum detection level of vaspin was found to be 25 nM by using this SPR-based sensor.

G protein-coupled-receptors (GPCRs) are a family of integral membrane proteins with seven transmembrane helices involved in a range of physiological processes, including cell signal transduction, sensory signaling, neuronal transmission and hormonal signaling. GPCRs are associated with almost every major therapeutic category or disease class, including pain, asthma, inflammation, obesity and cancer. More than 50% of all drugs target GPCRs and control their activities. Although many efforts have been given to use E. coli as host cell for the production of GPCRs, it is difficult to express because of their strong hydrophobicity and complicated structure. In this study, human parathyroid hormone receptor type 1, B class GPCR, was expressed in E. coli at high-level mainly as insoluble form after cloning of the gene into bacterial expressing vector, pDEST15. The expressed receptor was successfully solubilized with anionic detergent for further purification. This would be the first example for B class GPCR produced form E. coli in miligram quantity which is suitable amount for high-resolution structural techniques, such as solid-state NMR and crystallography. This B class GPCR production method can be applied to development of GPCR biosensor that might be wildly used in the field of diagnosis and drug discovery.