• Journal of Microbiology and Biotechnology
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• v.17 no.11
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• pp.1751-1757
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• 2007

Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli $DH5{\alpha}$ at $37^{\circ}C$ for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at $20^{\circ}C$ in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature ($T_t$) of levansucrase-ELP[V-40] was $17^{\circ}C$ with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification.

• Bulletin of the Korean Chemical Society
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• v.31 no.9
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• pp.2493-2496
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• 2010

Erythropoietin (EPO) is mainly produced in kidney and stimulates erythropoiesis. The use of recombinant EPOs for doping is prohibited because of its performance enhancing effect. This study investigated whether biosimilar EPOs could be differentiated from endogenous one by iso-electro-focusing plus double blotting and SDS-PAGE for antidoping analysis. The established method was validated with positive control urine. The band patterns were reproducible and meet the criteria, which was made by world anti doping agency (WADA). Isoelectric focusing was conducted in pH range 2 to 6. Recormon (La Roche), Aropotin (Kunwha), Epokine (CJ Pharm Co.), Eporon (Dong-A), Espogen (LG Life Sciences), and Dynepo (Shire Pharmaceuticals) were detected in basic region. All biosimilars showed discriminative isoelectric profiles from endogenous EPO profiles, but they showed different band patterns with the reference one except Epokine (CJ Pharm Co.). Next, SDS-PAGE of biosimilar EPOs resulted in different molecular weight patterns which were distributed higher than endogenous EPO. Commercial immune assay kit as an immune affinity purification tool and immobilized antibody coated magnetic bead were tested for the purification and concentration of EPO from urinary matrix. The antibody-coated magnetic bead gave better purification yield. The IEF plus double blotting and SDS-PAGE with immunoaffinity purification method established can be used to discriminate biosimilar EPOs from endogenous EPO.

• Journal of the Korean GEO-environmental Society
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• v.9 no.4
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• pp.77-84
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• 2008

Soil purification theory is the method using the soil micro-organism like aerobic and anaerobic for treatment of wastewater. The soil has many kinds of micro-organism and it multiply as change of the environment. Unlikely other methods, the soil purification theory is adaptable to inflow water change; moreover, it can process the T-N, T-P without any special method and management. The top is covered with the improved soil which can remove the bad smell and is used for resting place according to planting the lawn. This study is focused on analysis of the treatment processing of wastewater comparing inflow with outflow water. As a results, removal rate of the processing the BOD, COD and SS is almost 90~100% and it is 60~80% in T-N, T-P.

• Applied Biological Chemistry
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• v.11
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• pp.63-66
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• 1969

The purification methods of bacterial protease have been published by many workers, especially by the using of ion exchange resins. But in the practical application to obtain a comparatively purified enzyme, the known methods do not give always a satisfiable results. Here we developed an industrially applicable method for purification of bacterial protease with the using of tannin. By the adaptation of the optimal conditions of this method on the purification, a 150000 unit/g. (Fuld Gross unit) of protease sample could obtained.

• Entomological Research
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• v.48 no.6
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• pp.533-539
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• 2018

The RNA interference (RNAi) has been considered as an important genetic tool and applied to develop a new living modified (LM) crop trait which is an improvement of nutrient quality or pest management. The RNAi of DvSnf7 has been used for resistance to LM maize and the Western Corn Rootworm which is a major agricultural pest for the US Corn Belt. Most of the environmental risk assessments (ERA) of double strand RNA (dsRNA) have been performed using in vitro transcript products, and not in vivo expressed product. A large amount of dsRNA was required for the acute toxicity assay of water fleas. Therefore development of massive dsRNA purification techniques is critical. Daphnia, a freshwater microcrustacean, is a model organism for studying cellular and molecular mechanism involved in life history traits and ecotoxicology. In this study, we established the massive dsRNA purification method using Escherichia coli and implemented acute toxicity assays to Daphnia magna. As a result, the present RNase A and DNase I, dsRNA was efficiently purified without any special techniques or equipment. Even though purified dsRNA existed during the acute toxicity test, lethality or abnormal behavior were not observed in D. magna. These results indicated that GFP and DvSnf7 dsRNA were not significantly affected to D. magna due to their lack of sequence matching in its genome. The purification method of dsRNA and the acute toxicity assay of water fleas using purified dsRNA would be suitable for the toxicological studies of LMOs to aquatic non-target organisms.

• Journal of Ginseng Research
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• v.12 no.2
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• pp.164-172
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• 1988

The solvent separation step in the conventional method for quantitative analysis of ginsenosides was substituted by purification through a small reverse-phase $C_18$-column resulting in the decrease of analysis time by one fourth. New method showed high recovery of total ginsenosides but low recovery in protopanaxatriol-ginsenosides. Sugars did not affect the recovery by the amount in usual root sample. Coefficient of variation in recovery of ginsenosides was lower in the rapid minicolumn method. Optimum load of ginsenosides to minicolumn was 10 to 15 mg. The rapid minicolumn method showed highly significant correlation with the solvent separation method for dried root and red ginseng. For the rapid minicolumn method a small acryl device was used for the simultaneous extraction of 8 samples. This method appeared to be beneficial in cost and for the health of analyst.

• Korean Journal of Food Science and Technology
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• v.20 no.6
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• pp.780-786
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• 1988

In order to investigate the most efficient and rapid method for the purification of enterotoxin A from Staphylococcus aureus M 7/1, various methods such as ion-exchange chromatography on Amberlite, and CM-cellulose. gel filtration on Sephadex G-50, 75, 100 and Sephacryl, and fast protein liquid chromatography (FPLC) were applied and compared in terms of purity and speed. Although ion-exchange chromatography on Amberlite resin was good enough to remove other materials in culture medium from enterotoxin, and convenient, and fast method, the purity of this method was less than 70%. However. carboxymethyl ion-exchange column showed to be better purity than that of Amberlite method. The yields of these two methods were about 70% and 75%, respectively. When gel filtration methods on Sephadex G-50, 75, 100 and Sephacryl were applied, the purity was about 90%. Fast protein liquid chromatography was found to be the most efficient method in terms of purity (97%) and speed. The combined method, gel filtration after CM-cellulose column (stepwise elution) treatment can be also used as a efficient method particularly for the purification of large volume of sample.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.3 no.1
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• pp.27-32
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• 1970

A study on the purification of the surf clam Mactra Sulcataria REEVE for the remove of sand and fecal piles was conducted in the laboratory. The sand was mostly distributed around the inhalent and exhalent siphon, mantle and gill, and were also distributed in the digestive tract including the mid-gut gland. The sand particles in the digestive tract were extremely small and their sizes were about $180\times10\mu\;to\;550\times200\mu$. It could be seen that there was little, if any, difference in the rate of sand removal when either the hanging Purification method was used or the method of laying the surf clams in thick or thin layers on the bottom of the experimental vessel was used. The surf clam removed about $50\%$ of its sand during the first hour of purification. The weight of the removed sand and fecal piles on a dry basis reached a constant value after 22 to 26 hours of purification. After 42 hours of Purification, no sand could be seen in the fecal piles. The surf clam removed the sand in its body more rapidly when it was in sea water with the pH of 8.75 than when it was in natural sea water. Also high temperature had a much greater depressing effect upon the removal of sand In the surf clam than did natural sea water. Also low salinity had greater accelerating effect upon the removal of sand in the body than did natural sea water. However the surf clam died when the salinity was $8.19\%_{\circ}$.

• Journal of Microbiology and Biotechnology
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• v.18 no.10
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• pp.1648-1654
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• 2008

Superoxide dismutase (SOD) removes damaging reactive oxygen species from the cellular environment by catalyzing the dismutation of two superoxide radicals to hydrogen peroxide and oxygen. Extracellular superoxide dismutase (EC-SOD) is a tetramer and is present in the extracellular space and to a lesser extent in the extracellular fluids. Increasing therapeutic applications for recombinant human extracellular superoxide dismutase (rEC-SOD) has broadened interest in optimizing methods for its purification, with a native conformation of tetramer. We describe a solid phase refolding procedure that combines immobilized metal affinity chromatography (IMAC) and gel filtration chromatography in the purification of rEC-SOD from Escherichia coli. The purified rEC-SOD tetramer from the $Ni^{2+}$-column chromatography is refolded in Tris buffer. This method yields greater than 90% of the tetramer form. Greater than 99% purity is achieved with further purification over a Superose 12PC 3.2/30 column to obtain the tetramer and specific activities as determined via DCFHDA assay. The improved yield of rEC-SOD in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.

• Journal of Microbiology and Biotechnology
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• v.19 no.7
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• pp.727-733
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• 2009

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-n-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite,N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other Histagged proteins expressed in E. coli.