• Journal of Microbiology and Biotechnology
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• v.9 no.4
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• pp.386-392
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• 1999

A large-scale culture of hepatitis A virus in human diploid MRC-5 cells was conducted. In a roller bottle culture, the virus was grown to a maximum titer in 3 weeks after infection. Over 95％ of the cell-associated virus was excreted after culturing the infected cells in suspension media without fetal bovine serum for 3 days. The cultured virus was inactivated with formalin, concentrated by ultrafiltration, and partially purified by ultracentrifugation in a non-ionic gradient medium of Renocal. Two separate peak fractions showing high anti-HAY ELISA titer were pooled and about 40％ of HAV antigen was recovered by this purification procedure. Of the partially purified vaccine, the protein pattern in SDS-PAGE and immunogenicity in mice were compared with a commercial HAV vaccine. In SDS-PAGE, the purified vaccine in this study and the commercial vaccine showed almost the same protein pattern. The seroconversion rate of the purified vaccine in mice was not different from that of the commercial vaccine. Therefore, we could prepare a good grade of HAV vaccine by a simple purification procedure although the purification itself was not completed.

• Journal of Ginseng Research
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• v.44 no.6
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• pp.784-789
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• 2020

Background: The separation of isomeric compounds from a mixture is a recurring problem in chemistry and phytochemistry research. The purification of pharmacologically active ginsenoside Rb₃ from ginseng extracts is limited by the co-existence of its isomer Rb₂. The aim of the present study was to develop an enzymatic elimination-combined purification method to obtain pure Rb₃ from a mixture of isomers. Methods: To isolate Rb₃ from the isomeric mixture, a simple enzymatic selective elimination method was used. A ginsenoside-transforming glycoside hydrolase (Bgp2) was employed to selectively hydrolyze Rb₂ into ginsenoside Rd. Ginsenoside Rb₃ was then efficiently separated from the mixture using a traditional chromatographic method. Results: Chromatographic purification of Rb₃ was achieved using this novel enzymatic elimination-combined method, with 58.6-times higher yield and 13.1% less time than those of the traditional chromatographic method, with a lower minimum column length for purification. The novelty of this study was the use of a recombinant glycosidase for the selective elimination of the isomer. The isolated ginsenoside Rb₃ can be used in further pharmaceutical studies. Conclusions: Herein, we demonstrated a novel enzymatic elimination-combined purification method for the chromatographic purification of ginsenoside Rb₃. This method can also be applied to purify other isomeric glycoconjugates in mixtures.

• KSBB Journal
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• v.30 no.6
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• pp.291-295
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• 2015

The limited productivity of natural shell matrix proteins has hampered the investigation of their biochemical properties and practical applications, although biominerals in nature obtained by organic-inorganic assemblies have attractive mechanical and biological properties. Here, we prepared a vector for the expression of a fusion protein of a shell matrix protein from Pinctada fucata (named as GRP_BA) with the GRGDSP residue. The fusion protein of BA-RGD was simply produced in E. coli and purified through sequential steps including the treatment with $CaCl_2$ and EDTA solution for cell membrane washing, mechanical cell disruption and the application of non-ionic surfactant of Triton X-100 for BA-RGD inclusion body washing. The production yield was approximately 60 mg/L, any other protein band was not observed in SDS-PAGE and it was estimated that above 97% endotoxin was removed compared to the endotoxin level of whole cell. This study showed this simple and easy purification approach could be applied to the purification of BA-RGD fusion protein. It is expected that the protein could be utilized for the preparation of biominerals in practical aspects.

• 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.

• Journal of Radiation Protection and Research
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• v.48 no.2
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• pp.77-83
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• 2023

Background: The purpose of this study is to purify uranium (U[VI])-contaminated soil-flushing effluent using the precipitation-distillation process for clearance. Precipitation and distillation are commonly used techniques for water treatment. We propose using a combination of these methods for the simple and effective removal of U(VI) ions from soil-flushing effluents. In addition, the U concentration (Bq/g) of solid waste generated in the proposed treatment process was analyzed to confirm whether it satisfies the clearance level. Materials and Methods: Uranium-contaminated soil was decontaminated by soil-flushing using 0.5 M sulfuric acid. The soil-flushing effluent was treated with sodium hydroxide powder to precipitate U(VI) ions, and the remaining U(VI) ions were removed by phosphate addition. The effluent from which U(VI) ions were removed was distilled for reuse as a soil-flushing eluent. Results and Discussion: The purification method using the precipitation-distillation process proposed in this study effectively removes U(VI) ions from U-contaminated soil-flushing effluent. In addition, most of the solid waste generated in the purification process satisfied the clearance level. Conclusion: The proposed purification process is considered to have potential as a soil-flushing effluent treatment method to reduce the amount of radioactive waste generated.

• Journal of Microbiology
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• v.45 no.6
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• pp.593-596
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• 2007

In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.

• Textile Coloration and Finishing
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• v.17 no.4 s.83
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• pp.21-26
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• 2005

A combination separation system is composed of three parts, simple microfiltration unit for the pretreatment of real waste IPA, pervaporation unit with plate and frame type module(the effective membrane area 9,040$cm^2$), and simple ultrafiltration unit as a refiner. Utrafiltration module with hollow fiber membrane(MWCO 10,000) used to purify waste aqueous IPA solution. In addition, the flux of $CMPA-K^+$ composite membrane for waste aqueous IPA solution was very steady-state with long experiment time(30 days). And the standard deviation($\sigma$) was 0.152 and then the coefficient of variation($CV\%$)was 10.82 The IPA concentration on the membrane performance using pervaporation module system could be increased from $89.85wt(\%)$ to more than $99.90wt\%$ in about 8hr at operation temperature of $70^{\circ}C$ using the pervaporation module system. Therefore, a combination separation process system of simple filtration and pervaporation was very effective for the purpose of the IPA purification and reuse front industrial electronic components cleaning process.

• Journal of Industrial Technology
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• v.36
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• pp.33-37
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• 2016

Cellulose binding domains (CBDs) of cellulases are thought to assist in the hydrolysis of insoluble crystalline cellulose. To gain sufficient amount of CBDs, the self-cleavable intein tag was used for expression and purification of Trichoderma reesei cellobiohydrolase I CBD in E. coli. Synthetic CBD genes, CBD or linker-CBD were cloned into expression vector pTYB11. Recombinant CBDs were successfully purified by intein mediated purification with an affinity chitin-binding domain. The final yields of recombinant CBD and linker-CBD were 3.2 mg/L and 1.4 mg/L, respectively. The functional bindings of recombinant CBDs were confirmed by Avicel binding experiments. The simple and easy purification method using self-cleavable intein tag can be further used in pretreatment of crystalline cellulose or characterization of engineered CBDs.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.351-354
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• 2005

A new isolation and purification method was developed aiming at increasing yield and purity for homoharringtonine. This method was a simple andefficient procedures, for the isolation and purification of homoharringtonine from Cephalotaxus koreana, consisting of solvent extraction, adsorbent treatment, low-pressure chromatography, and high performance liquid chromatography (HPLC). The crude homoharringtonine was efficiently pre-purified adequately to perform HPLC through a combination withadsorbent treatment and low-pressure chromatogaphy. The homoharringtonine can be simply obtained with high purity and yield from crude homoharringtonine by HPLC. Purified homoharringtonine was characterized.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.02a
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• pp.87-87
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• 2013

Sample concentration and purification processes are essential in the bio-analytical and pharmaceutical fields because most bio samples or media are extremely sophisticated. To concentrate and purify specific substances, passive membrane type filters have been utilized, which is driven by size or charge differences between target and others. The traditional and representative method to identify nucleic acid sequences in the complex biosample is gel electrophoresis, which has been worked by size and net charge of molecules. The adsorption phenomena have been also utilized to concentrate and purify biomolecules. This adsorption of biomolecule can be controlled under specific salts and surfaces as well as surface area. To utilize the differences of physical properties of molecules or bio-targets such as virus, bacteria, and cells, the nanotechnologies can be introduced in target concentration, purification, and isolation processes. In here, I'd like to briefly survey typical examples of nanobiotechnologies which are introduced in sample treatment. Also I specifically demonstrate two different simple techniques to concentrate and detect bacteria from the samples using multifunctional silica nanotube (SNT).