• 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.12 no.6
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• pp.916-920
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• 2002

This report describes a high-level expression of human alpha-2a interferon ($IFN{\alpha}-2a$) in Escherichia coli and its pilot scale purification by using a monoclonal antibody-independent chromatographic procedure that is based on anion-exchange, cation-exchange, hydrophobic interaction, and gel filtration. The recombinant E. coli produced much more $IFN{\alpha}-2a$ in a soluble form, when cultivated at low temperatures than at high-temperature fermentation. However, if the bacterial growth was taken into consideration, fermentation at $30^{\circ}C$ seemed optimal for the interferon production. By using our new protocol, we recovered approximately 160 mg of $IFN{\alpha}-2a$ with a specific activity of $3.59{\times}10^8$ IU/mg from 201 of the broth. The gel permeation chromatographic and SDS-PAGE indicated that the interferon preparation was purified to homogeneity and was of the correctly folded fast-migrating monomer.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.6
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• pp.423-427
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• 2004

A simple purification procedure of bioactive human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture has previously been described. In this study the protein was purified to apparent homogeneity with an overall yield of $80.1\%$ by ammonium sulfate precipitation and a single chromatographic step involving FPLCanion exchange chromatography. The purified hGM-CSF revealed at least five glycosylated forms ranging from $21.5{\~}29$ kDa, and its biological activity was independent of the glycosylation pattern. This is the first purification report of recombinant hGM-CSF to apparent homogeneity from rice cell suspension cultures.

• Journal of Life Science
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• v.19 no.5
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• pp.561-565
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• 2009

Human Tissue factor is an essential enzyme activator that forms a catalytic complex with factor VII/ VIIa, and catalyzes both the extrinsic and intrinsic blood coagulation cascades. The extracellular domain of human tissue factor is responsible for association with the biological partner. The efficient procedures for preparing biologically active human tissue factor are essential for the preclinical and clinical studies with coaguligands. An expression vector in Escherichia coli has been constructed to direct the production of extracellular human tissue factor without a fusion protein or a $His_6$ at the N-terminus. The recombinant human tissue factor was expressed in large amounts as a non-native state in E. coli. The recombinant protein was simply renatured during the DEAE-sephacel chromatographic purification procedure. Our expression and purification system does not require a protease treatment or an additional chromatographic step to remove a fusion contaminant, which provides a very useful alternative to conventional expression systems for the production of human tissue factor.

• Journal of Radiopharmaceuticals and Molecular Probes
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• v.2 no.2
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• pp.84-95
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• 2016

Considerably increasing interest in using the theranostic isotopes/ isotope pairs of radiometals like $^{44/47}Sc$ and $^{64/67}Cu$ for diagnosis and/or therapeutic applications in the nuclear medicine procedures necessitates its reliable production and supply. Separation and purification of no-carrier-added (NCA) isotopes from macro quantitates of the irradiated target matrix along with other impurities is a cardinal procedure amongst several other steps involved in its production. Multitudinous methods including but not limited to liquid-liquid (solvent) extraction, extraction chromatography (EXC), ion exchange, electrodeposition and sublimation are routinely applied either solitarily or in combination for the separation and purification of radioisotopes depending on their production routes, radioisotope of interest and impurities involved. However, application of EXC though has shown promises towards the numerous separation techniques have not received much attention as far as its application prospects in the field of nuclear medicine are concerned. Advances in the recent past for application of the EXC resins in separation and purification of the several medically important radioisotopes at ultra-high purity have shown promising behavior with respect to their operation simplicity, acidic and radiolytic stability, separation efficiencies and speedy procedures with the enhanced and excellent extraction abilities. In this mini review we will be talking about the recent developments in the application and the use of EXC techniques for the separation and purification of $^{44/47}Sc$ and $^{64/67}Cu$ for medical applications. Furthermore, we will also discuss the scientific and practical aspects of EXC in the view of separation of the NCA trace amount of radionuclides.

• Journal of Food Hygiene and Safety
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• v.9 no.2
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• pp.89-94
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• 1994

A simple, safe, and sensitive gas chromatographic method using packed column and electron capture detector to analyze 2, 4-D herbicide in imported lemon, grapefruit, and orange was described and its usefulness evaluated. In this scheme of analysis the acid herbicide was converted into its alkyl esters by an one-step reaction prior to analysis. The herbicide in the fruits was extracted with ethyl acetate and partitioned against dichloromethane for purification, and the extracts finished partitioning were derivatized with alcohol, using sulfuric acid as a catalyst to form the corresponding alkyl derivatives. The analytical scheme studiedwas found to be applicable for the herbicide in the fruits without a column clean-up procedure. The mean recoveries of the herbicide for lemon samples fortified at 0.1 mg/kg and 1.0 mg/kg were 93% and 95%, respectively. The detection limit was 0.5 $\mu\textrm{g}$/kg for 2.4-D methyl ester.

• The Korean Journal of Zoology
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• v.33 no.1
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• pp.119-125
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• 1990

A Potent inhibitor of trypsin and other various serine proteases including chymotrypsin, elastase, kallikrein, plasmin and subtilisin, has been purified to homogeneity from chick skeletal muscle by convendonal chromatographic procedures. The Inhibitor has an apparent molecular weight of 66, 000 dalton as determined by gel filtration. When the purified inhibitor was electrophoresed in the presence of sodium dodecyl sulfate, there appeared rwo protein bands having molecular weights of 66, 000 and 64, 000 dalton. The 64, 000 dalton protein seems to be the product of 66, 000 dalton protein by a lin'ited proteolysis during the purification procedure or in viuo. Thus, it seems to consist of a single polypeptide. The inhibitor appeared to be glycoprotein and have an isoelectric point of 7.4. It contains relatively large amount (8.33 mole%) of cysteine residues.

• International Journal of Industrial Entomology and Biomaterials
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• v.37 no.2
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• pp.95-99
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• 2018

In insect defense system, antimicrobial peptides (AMPs) are one of important biological molecules to survive in a variety of environments. Insect can synthesize AMPs to protect against invading pathogens in humoral immune response. Taking more advantage of biological antimicrobial molecules, we report antibacterial activity of two cecropin type peptides, cecropin and moricin, isolated from the silkworm against four salmonella species. In this work, we purified antimicrobial candidate peptides (AMCP) from the extracts of immune challenged silkworm larval hemolymph by two-step chromatographic purification procedure, cation exchange and gel permeation chromatography. The molecular weights of purified peptides were estimated to be about 4 ~ 5 kDa by Tricin SDS-PAGE analysis, and identified as silkworm cecropin and moricin by NCBI BLAST homology search with their N-terminal amino acid sequences. As antibacterial activity assay, the purified peptides showed stronger antibacterial activity against Salmonella pathogens with an MIC value of $1{\sim}4{\mu}g/mL$. Therefore two cecropin type peptides purified from the silkworm will be valuable potential materials for development of new natural antibiotics.

• Bulletin of the Korean Chemical Society
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• v.24 no.4
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• pp.413-420
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• 2003

A gas chromatography/mass spectrometric assay method was developed for the simultaneous determination of neutral and bacis twenty-five disruptors $(ED_S)$ in frog and fish. Afther homogenization and sonication of 5 g of sample, purification was achieves in one step with a solid phase extraction procedure using silica gelflorisl. Eluton was performed with 50mL of acetone : n-hexane (1 : 9) solution. The eluate was concentrated to approximately 10uL and dissolves with 100 uL of hexane and analyzed by GC-MS (SIM). The peaks had good chromatographic properties and the extraction of these compounds from sample also gave relatively high recoveries with small variatoins. Detection limits were 0.1 ng/g for 4-nitrotoluene, benzophenone, hexachlorobenzene, atrazine, malathion, o,p-DDT, o,p-DDT and permethrin, and 0.2 ng/g for heptachlor epoxide, γ-chlordane, α-chlordane, p,p'-DDE, p,p'-DDD, cypermethrin and fenvalerate, and 0.3 ng/g for trifluralin, metribuzin, alachlor, dieldrin and p,p'-DDT, and 0.5 ng/g for heptachlor, aldrin and parathion, and 0.7 ng/g for endrin, and 0.8 ng/g for nitrofen. The recoveries were between 33 and 109%. The method was used to analyze twenty-five frogs and forty-six fishes fishes samples caught from various regions in Korea. Benzophenone was detected at concentration of up to 17.2 ng/g in frog or fish. Heptachlor, aldrin, γ-chlordane, p,p'-DDE, p,p'-DDD, endrin and o,p-DDD were detected at concentrations of 0.7-12.5 ng/g in frog or fish. Also significant leveles of dieldrin (up to 22.5 ng/g) were observed. The developed method may be valuable to be used to the national monitoring project of EDS in biota samples.

• Journal of Microbiology and Biotechnology
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• v.27 no.7
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• pp.1281-1287
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• 2017

Bone morphogenetic protein-4 (BMP-4) is considered to have therapeutic potential for various diseases, including cancers; however, the high expression of biologically active recombinant human BMP-4 (rhBMP-4) needed for its manufacture for therapeutic purposes has yet to be established. In the current study, we established a recombinant Chinese hamster ovary (rCHO) cell line overexpressing rhBMP-4 as well as a production process using 7.5-l bioreactor (5 L working volume). The expression of the mature rhBMP-4 was significantly enhanced by recombinant furin expression. The combination of a chemically defined medium and a nutrient supplement solution for high expression of rhBMP-4 was selected and used for bioreactor cultures. The 11-day fed-batch cultures of the established rhBMP-4-expressing rCHO cells in the 7.5-L bioreactor produced approximately 32 mg/l of rhBMP-4. The mature rhBMP-4 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure, resulting in a recovery rate of approximately 55% and a protein purity greater than 95%. The N-terminal amino acid sequences and N-linked glycosylation of the purified rhBMP-4 were confirmed by N-terminal sequencing and de-N-glycosylation analysis, respectively. The mature purified rhBMP-4 has been proved to be functionally active, with an effective dose concentration of $EC_{50}$ of 2.93 ng/ml.