Earticle

The thiol-disulfide oxidoreductase thioredoxin-1 (TRX) is known to be secreted by leukocytes and to exhibit cytokine-like properties. Extracellular effects of TRX require a functional active site, suggesting a redox-based mechanism of action. However, specific cell surface proteins and pathways coupling extracellular TRX redox activity to cellular responses have not been identified so far. We first found that exogenous TRX not only scavenger but also effected intracellular proteins using proteomic approaches. Therefore, the effect of a thiol-antioxidant protein TRX have appropriated on regulation in a human skin malignant melanoma cell line (A375). Mechanism-based His pull-down and immunoprecipitation technique used to identify TRX binding partner proteins. We found that up/down regulated intracellular proteins were identified under the condition of LPS-treated A375 cells and co-treat with exogenous TRX using in vitro 2-DE proteomic approaches. As well as, confirmed in vivo mouse inflammation models used to quantitatively measure IL-6, IL-10, MCP-1 and TNF protein levels in serum samples. Our studies demonstrate that exogenous TRX has anti-inflammatory properties and intracellular regulator activity in vivo and in vitro. These results have a therapeutic role in skin inflammation therapy.

Anti-angiogenesis was carried out for investigating of anti-cancer and anti-obesity mechanism from natural products such as Anthrisci Radix, Psoraleae Semen. The formation of angiogenesis was completely inhibited by these 2 kinds of natural products. The maximum safe dose of Anthrisci Radix, Psoraleae Semen is 0.2 ppm and 10 ppm, and the fraction of these natural products also showed higher anti-angiogenic effect, F5 and F2 fraction, respectively. F5 fraction of Anthrisci Radix completely surppressed angiogenesis with incresing its dose, while F2 fraction of Psoraleae Semen inhibited angiogenesis at 10 ppm. These 2 kind of natural products will be a good anti-angiogenic substances for anti-cancer and anti-obesity.

The development of environmentally responsive drug carriers requires a new method for assembling stimuli-responsive hydrogel. Recent progress suggests that short peptide motifs can be engineered into biopolymers with specific temperture-dependent behavior.Protein polymers derived from thermally responsive elastin-mimetic peptide can be synthesized with near-absolute control of macromolecular architecture using genetic engineering techniques. Elastin-mimetic triblock copolymers contain hydrophobic and hydrophilic segments and display different phase behavior in aqueous solution by changing the copolymer's length or sequence. The selective collapse of the hydrophobic blocks above the transition temperature can cause 3 dimensional protein-based nanoparticles or nano-textured hydrogels. We designed triblock peptide copolymers with various length and are investigating their expression in E. coli.

Separation and/or removal of RNA and endotoxins, the major contaminants found in alkaline cell lysate in plasmid DNA (pDNA) purification process, has been cumbersome due to their similar properties (mostly negative charge) to pDNA. In our previous study, differential affinity of pDNA, RNA and endotoxins towards various metal ions was demonstrated that endotoxins and RNA preferentially and strongly bind to Cu2+-IDA and that pDNA binds only to Fe3+-IDA in the solution lacking RNA and endotoxins. By harnessing these preferential affinities in a single stage monolith-based immobilized metal affinity chromatography (IMAC) comprising Cu2+-IDA followed by Fe3+-IDA columns in series, pDNA was recovered in pure form at a high yield from the flowthrough from the Cu2+-IDA column where RNA and endotoxins were selectively captured. Furthermore, the proposed processing scheme, empowered by the advantages of monolith matrix (e.g. high flowrate and/or throughput, short processing time), exhibited significantly higher productivity over the conventional chromatographic processing schemes using particle based supports, facilitating the scale-up of the developed process to an industrial scale. Additional polishing step to remove genomic DNA and/or pDNA isoforms (e.g. linear and open circular forms) from supercoiled pDNA gave pharmaceutical-grade pDNA capable of addressing regulatory specifications, thereby achieving significant intensification of pDNA purification process in an economically viable way.

The analysis of bases is important in many biotechnology research area. The bases are the building block of nucleotide and bases change of structure and function is the main causes of inherited diseases and human cancers. Separation investigations for DNA focus on the base pairs guanine and cytosine in nucleotide This study of continuos separation technology by SMB is expected to have a large effect on the understanding nucleotide knowledge as well as on producing nucleotides quantitatively.Guanine and cytosine separation was studied by HPLC (High Performance Liquid Chromatography) simulations with a change of sample concentration and eluent flow rate. Cytosine and guanine of high-purity were simulated by the 3 - zone Aspen simulator. The separation performance of aspen simulation and the effect of operating parameters, such as extract flow rates, switching time, feed flow rate and feed concentration, were studied under various operating conditions. Aspen simulation leading to complete separation were observed and explained theoretically.The SMB experiment conditions were with out a feed concentration of 0.1 mg/ml, and by changing switching time. As a result, the highest raffinate cytosine purity was 98.4% and the extract guanine purity was 97.9%.

Streptococcus pneumoniae causes many types of pneumococcal infection; especially, the incidence of pneumococcal disease which is the highest in infants under 2 years and in people over 60 years of age. Capsular polysaccharides (CPS) are the primary basis for pathogenicity, antigenic and serotypes. To purify CPS for producing vaccine, we developed a simple process including four steps. Initially, the cells were collected by centrifugation. And they were lysed using detergent. Second, the proteins and cell debris were precipitated and removed by the addition of acetic acid. Third, pure CPS was prepared by ethanol concentration fractionation (50~80%, v/v) at pH 7.0. Finally, the purified CPS was further dialyzed against dH2O. This process would save cost, time and labor in comparison with current methods developed by others. Thus, this modified process would be suitable for preparation of conjugated CPS as vaccine to reduce the pneumococcal diseases.

Recently chicken egg yolk has been considered as an ideal alternative antibody source. The production for antibody in eggs and the extraction of spectific antibody from egg yolk are increasingly attracting the interest of the sxientific community. Although some of the separated egg yolk components which are water, lipoprotein and protein are already commercialized, the separation efforts are limited to a pure component.IgY has Particularly a equivalent function with IgG which is in serum. This study was carried out to separate and analyze IgY from chicken egg. To perform this experiment, egg yolk powder diluted with cold water was centrifuged after adjusting the pH to 5.0. 0.01% carrageenan was added to reduce the lipid. The supernatant was filtered through a Whatman no. 1 filter paper. The concentrated sample was loaded onto a column equilibrated with 20 mM phosphate buffer at pH 7.0 and eluted with phosphate buffer and 1M NaCl. IgY was analytically separated by using ion exchange, gel filtration and high pressure liquid chromatography. By changing the amount of egg yolk powder, eluted height of peaks were quantitatively changed.

Continuous use of fossil fuels is now widely recognized as unsustainable because the burning of an enormous amount of fossil fuel has increased the CO₂level in the atmosphere, contributing to the greenhouse effect. Biomass has been focused on as an alternative energy source. Among biomass, microalgae have a higher photosynthetic efficiency than other biomass. In addition, microalgae have been promoted as a future source of transportation fuels primarily because of their stated potential to produce up to 10 times the amounts of oil per acre than vegetable oils, animal fats and crops. In this study, lipid compounds were extracted by supercritical carbon dioxide from microalgae biomass and their fatty acid composition was analyzed. HPLC with an evaporative light scattering detector and GC with a flame ionization detector were used to identify lipid families and fatty acids, respectively. The main aim of this work is to investigate the kinetics of the supercritical fluid extraction and the effect of operating condition on the extraction yield and on the fatty acid composition of lipid extracts.

The solvent treatment of paclitaxel 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 inrelatively 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. Thus, polarity of the solvent was an important factor governing the morphology of the paclitaxel. 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 5.85wt%, 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.

Various strains of bacteria have been found to produce the cellulose having similar chemical structure to that of plant cellulose but different physical and chemica properties. In fact the bacterial cellulose (BC) has much improved properties, regarding its mechanical strength, biocompatibility, water holding capacity, degree of polymerization and ultra fine and highly pure fiber network. However the dilemma of its quantitative production is still a hot issue. Massive efforts have been made to enhance the BC production so as to make it commercially encouraging and better substitute of plant cellulose. Besides investigating the new and cheaper carbon sources for BC production one additional approach presently in progression is the addition of supplementary additives to enhance the BC production. Glucuronic acids are notably involved in the biochemical pathway of bacterial cellulose production. In our previous studies the use of glucuronic acid oligomers as an additional substrate showed positive effect on the BC production. In present study glucuronic acid monomers were used to find their effect on the production of BC and Water soluble oligosaccharides (WSOS). Experimental results show that BC production in both static and shaking cultures was increased to a great extent with the addition of 0.5 ~ 4.0% glucuronic acid monomers. Maximum of 2 and 1.5 times production of BC and WSOS respectively have been achieved compared to control after 10 days in both shaking and static cultures

The Gram-positive pathogenic bacterium Streptococcus pneumoniae is cause of pneumonia, meningitis, otitis media and sepsis [1]. In particular, more than 90% pneumonia patients catch a pneumonia disease due to S. pneumoniae. Its elements of the capsular polysaccharide, C substance, F antigen and M protein are the antigens. That of the capsular polysaccharide is used to product of vaccines. 91 kinds of capsular polysaccharides types are known until now [2]. In this study, we obtained capsular polysaccharides from S. pneuminae type 19A (CPS19A). CPS19A was purified by chemical treatment and ethanol precipitation. The size of capsular polysaccharide was reduced by acid hydrolysis and then hydrolysate of CPS19A was purified by ethanol precipitation and gel permeation chromatography. Different size of CPS19A was carried out conjugation with the flagellin. The conjugation result was identified by thin layer chromatography, SDS-PAGE and western blot analysis.

In this study, Nanofiltration (NF) with TFC-SR2 membrane was used to recover ammonium lactate from model solution. A modeling method based on multistage version of NF process has been proposed to represent this process. Prior to process modeling, the structural and electrostatic parameters of the membrane used were determined by using the Spigler-Kedem (SK) model and steric hindrance pore model. For the estimation of model parameters associated with the NF process under consideration, experiments at different concentrations and operating pressures were performed. The lactate rejection at the given pressure was empirically represented as a function of its concentration by nonlinear curve fitting. For the determination of solution flux, the reflection coefficient and effective volume charge density were calculated by using the SK model together with the Teorell–Meyer–Sievers model. Time profiles of lactate concentration in the permeate predicted by the proposed model were in a good agreement with the experimental data. The utilization of the model for simulation study will be addressed, to select optimal process options and to optimize process conditions with consideration of the lactate yield and the accumulation of major impurities.

Proteins have wide applications in the medical, industrial and agricultural fields. They are usually produced in huge quantities in host cells such as Escherichia coli. However, high-level expression of recombinant proteins in E. coli often results in accumulation of insoluble aggregates known as inclusion bodies (IBs), thus requiring further solubilization, refolding and purification procedures to achieve functionally active products. Molecular chaperones have been applied successfully to refold various proteins both in vivo and in vitro, opening a new era in protein refolding. However, the exact function of individual molecular chaperones and the interaction between target protein and molecular chaperones are still unclear. We demonstrated that refolding cocktail comprising ClpB/DnaKJE with ATP regeneration system and a refolding additive, PEG, could significantly enhance the refolding efficiency of heat-denatured malate dehyrogenase (MDH). To further clarify the individual or synergistic roles of each chaperone, various molecular chaperones including His-ClpB, His-DnaK, His-DnaJ, His-GrpE, His-Gro EL, His-Gro ES, and His-Trigger Factor were recently cloned, expressed and purified, respectively. By using different temperature for heat-denaturation of MDH, protein aggregates of varying size were produced. Following the characterization of these protein aggregates (e.g. susceptibility to proteolysis and kinetics of solubilization), chaperoning efficiencies of the His-tagged molecular chaperones on the renaturation of the protein aggregates are being studied systematically. It is expected that our study will provide a better understanding of chaperone-assisted refolding process, thereby facilitating engineering implementation of a novel refolding strategy, folding-like-refolding approach based on refolding cocktail.

Commercial production of microalgal biomass requires several processes for recovery of biomass after large-scale cultivation. Harvesting of biomass requires a few solid-liquid separation steps. Since the broth of microalgae culture was generally dilute, it was needed to remove large volume of water from the broth. Hence, the downstream recovery process of microalgal biomass can be more expensive than the large scale cultivation process. In most cases, many harvest methods were used and claimed to contribute 20-30% to the total cost of biomass production. Microalgal biomass can be harvested by centrifugation, filtration or in some cases, sedimentation. In this study, we used state-of-the-art membrane filtration method (forward osmosis) for removal of water from cultivated broth of microalgae. Osmosis is a natural equilibrium process, therefore water passes from broth of microalgae culture to outside by diffusion until the salt concentration on both sides of the membrane are equal. If we used microalgal biomass as a raw material to produce biodiesel, moisture in the biomass would influence the economics of products. Because general dehydration method, thermal drying was more expensive than mechanical dewatering, filtration method was used prior to thermal drying.

Cellulose produced by certain strains of bacteria is known as bacterial cellulose (BC). BC is a high value commodity with specific applications and usage. For BC production the proper medium conditions and availability of carbon sources are of prime importance. Usually glucose is used as a main carbon source for BC production. However, besides glucose some other additional carbon sources may also be used. Water soluble glucuronic acid oligomers (WSOS) are produced as a by-product during BC production. WSOS are rich source of utilizable carbon. Hence, in present study the BC production by Acetobacter sp. in shaking and static cultures was conducted using basal medium with different amounts of WSOS (0 ~ 4.0%). The pH, amounts of BC, WSOS and glucose were determined after five and ten days of cultivation. It was found that in basal media containing up to 2.0 % of WSOS the amount of BC was almost 1.5 to 2 times higher compared to control. However, media contained the higher amount of WSOS (more than 2.0%) showed a smaller glucose consumption, lower pH and a lower production of BC. The glucose amount in the media was almost diminished after five days of cultivation while the BC amount and pH of the medium was continuously increased with increase in cultivation time. It was observed that in all of the WSOS added media the reduction in WSOS amount occurred which indicated its consumption during the process. From this whole study, it was inferred that using WSOS as an additional substrate worked as a nutrient after glucose exhaustion.

This paper presents the amino acid purification process showing how the filtration time is reduced by the use of an electric field. The transmembrane pressure (TMP) was reduced by 20 % in a particular electric field, the hydraulic filtration using an electric field provides an another substitute to the crossflow filtration in the purification of protein. The aim of the presented work is to investigate the recovery of dissolved amino acid using an microfiltration apparatus with an electric field. The results were compared with a focus on measuring the enhancement of the filtrate flux with the application of an electric field. Hydraulic microfiltration using an electric field was found to significantly improve filtration and serves as an interesting alternative to cross-flow filtration for the purification of amino acid. The conventional filtration theory was also used in order to determine the permeate rate and the resistance of the fouling-layer in the membrane. From the experimental results, the transmembrane pressure (TMP) was reduced by 20 % as the electric field increased. The resistance (Rm) of the membrane process in an electric field was reduced by over 200 % in comparison with an no-electric field.

The purification of protein has been widely used in the electrodialysis, ultrafiltration, microfiltration when comparing with the number of studies on the use of conventional membrane for different proteins’ separation [1~2]. But they were not used as a purpose of enhancing the permeability. The newly developed techniques superimpose additional forces such as the pressured hydraulic force. However, the principle disadvantage of this technique is that cannot be separated in high concentration of protein. In this paper a superimposed another force, that is, an electric field induces as a force on charged protein in order to reduce the surface layer on the membrane. A newly developed type by an electric force has been invented a electro-microfiltration membrane [3~5]. Present studies in this article are experimentally to test the performance of new-typed electro-microfiltration membrane at a low hydraulic pressure and induces high amount of permeates. As such, the membrane filtration was investigated based on the membrane properties measured experimentally using the cake filtration theory [6]. The microfiltration technique was chosen to reduce the transmembrane pressure (TMP) and to increase the permeate flux(see below figures). This study shows that this membrane system manufactured by a electro-microfiltration technique offers significant advantages. The purification of protein in this presented work is to investigate in the membrane process under the influence of an electric field. This paper presents with example from membrane process showing how the filtration time is reduced by the use of an electric field. The transmembrane pressure (TMP) was reduced by 20 % as the electric field increased. The concentration of protein in the membrane process in the presence of an electric field was reduced by over 300 % in comparison with the membrane process without an electric field. The hydraulic electrofiltration provides an another substitute to the crossflow filtration in the purification of protein.