• Korean Journal of Food Science and Technology
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• v.28 no.1
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• pp.184-189
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• 1996

A scavenger of superoxide anion radical which causes oxygen toxicity was isolated from Capsella bursa-pastoris, and its physicochemical properties were investigated. The scavenger was isolated and purified by solvent fractionation and liquid column chromatographies (Amberlite XAD-2, Sephadex LH-20, Bio gel P-2, ODS (silica gel with 100% octadecyl silanization)). An active compound of 0.25 g was finally isolated by Fast Protein Liquid chromatography (FPLC) from 100 g ethanol extract of Capsella bursa-pastoris. A 50% decrease of superoxide anion radical was obtained with the scavenger compound of 0.58 g. The compound was assumed to be a phenolic glycoside from its physicochemical properties.

• Animal cells and systems
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• v.1 no.3
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• pp.457-462
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• 1997

Antibacterial activity was induced in the haemolymph of the common cutworm, Spodoptera litura by the artificial injection of E, coli Ek132. Antibacterial peptides were purified from the immunized haemolymph by heat treatment, ion-exchange chromatography, gel filtration chromatography, and reverse phase FPLC, and their physicochemical characteristics were investigated. These purified antibacterial peptides designated as spodopsin la and Ib were named after Spodoptera litura. Spodopsin la and Ib had the apparent molecular masses of 3, 823 Da and 3, 886 Da, respectively, and about 20% of the sequences had basic amino acids, such as lysine and arginine but no cysteine. Also, spodopsin l was confirmed to be a new member of cecropin family having a similar amino acid sequence to cecropin of lepidopteran insects, such as Bombyx mori and Hyalophora cecropia. The purified spodopsin was active against gram-positive as well as gram-negative bacteria.

• Journal of Life Science
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• v.8 no.3
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• pp.326-332
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• 1998

Cyclodextrin glucanotransferase from B. stearothermophilus KJ16 that can produce both cyclodextrin glucanotransferase and cyclodextrinase was purified by ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-100 chromatography, and FPLC. The molecular weight of the purifice enzyme was about 65,000 dalton by SDS-PAGE. The optimal pH and temperature were 6.0 and $60^{\circ}C$, respectively. The enzyme was stable at $50^{\circ}C$ for 1 hr and in the pH range of 5.5 and 8.5. Mercaptoethanol and dithiothreitol inhibited the enzyme activity strongly. The enzyme produced 60% cyclodextrin(CD) from 5% soluble starch with the $^{\alpha}$, $^{\beta}$, $^{\gamma}$-CD ratio of 42:46:12. Amylopectin was the most suitable substrate with 67% conversion to CD.

• Journal of Microbiology and Biotechnology
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• v.6 no.1
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• pp.26-29
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• 1996

The genes coding for the urease of alkalophilic Bacillus pasteurii have been previously cloned and recently sequenced. (You, J. H., B. H. Song, J. H. Kim, M. H. Lee, and S. D. Kim (1995) Molecules and Cells 5, 359-369.) The recombinant Bacillus pasteurii urease expressed in an E. coli HB101 strain was purified 31.2 fold by using combinations of anion-exchange and hydrophobic chromatography followed by Mono-Q chromatography on a FPLC. In spite of the presence of three discrete structural peptide genes in the Bacillus pasteurii urease gene cluster, only one or two enzyme subunits have been observed to date. Here we report for the first time that the recombinant Bacillus pasteurii urease expressed in a E. coli strain consists of three distinct subunits. One large subunit was estimated to be of $M_r$=65, 200 and the two small-subunit peptides are of $M_r$=14, 500 and $M_r$=13, 700, respectively.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.28 no.6
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• pp.728-735
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• 1995

The peptidyl prolyl cis-trans isomerase(PPlase, EC 5.2.2.8) from Bacillus stearothermophilus SIC1 was extracted from the cells treated with by lysozyme. PPlase was purified from the cell extracts by heat treatment, ammonium sulfate precipitation, ion exchange chromatography and finally gel filtration (FPLC). The purity of purified the enzyme after Superose 12 column chromatography was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The molecular weight of the purified PPlase was estimated as 18,000 by SDS-PAGE. The 39 amino acid residues from the N-terminus were determined by the protein sequencer. The enzyme showed the optimum pH at 8.0 and was stable at the range of pH 7.0 to 8.0. The enzyme was considerably stable after heat treatment at $60^{\circ}C$ for 30 minutes, and the enzyme was quite stable up to $65^{\circ}C$. The presence of the PPlase in the refolding solution accelerated the isomerization rate of the assay peptide.

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.255-259
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• 1999

A 6.9-kb DNA fragment including the minimal Bacillus pasteurii urease gene cluster was subcloned into a high-copy-number plasmid vector, pUC19, and the recombinant B. pasteurii urease was overexpressed in Escherichia coli. The recombinant urease was purified 25.9-fold by using combinations of anion-exchange and gel-filtration chromatography followed by Mono-Q chromatography on a FPLC. N-terminal peptide sequencing analyses revealed that two distinct smaller peptide bands resolved on a 10-18％ gradient SDS-PAGE corresponded to UreA and UreB peptides, respectively. It was also shown that the ureB gene was translated from a GUG codon and the first methionine residue was post-translationally cleaved off. The native molecular weight of the recombinant urease was 176,000 and 2 nickel atoms were present per catalytic unit. pH stability studies of the purified enzyme showed that the recombinant Bacillus pasteurii urease is stable in alkaline pH range, which is similar to the enzyme of the evolutionarily related bacterium, Sporosarcina ureae.

• Animal cells and systems
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• v.2 no.3
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• pp.367-370
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• 1998

Two molecular species of apolipophorin-III (spoLp-III) were purified from the last instar larval hemolymph of Galleria mellonella by gel permeation chromatography (Sephadex G-100), ion exchange chromatography (DE-52), heat treatment (90C for 30 min) and Mono S FPLC, and were named apoLp-III-a and apoLp-lll-b, respectively. They were indistinguishable by SDS-PAGE but could be separated by native PAGE. The molecular mass of apoLp-III determined by SDS-PAGE was approximately 18 kDa. The N-terminal amino acid sequence of apoLp-III-b revealed high similarities with the apoLp-III from Manduca sexta.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2003.04a
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• pp.80-80
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• 2003

The isolation and purification of ferritin from the cricket, Gryllus bimaculatus haemolymph were accomplished by anion exchange column chromatography using HiTrap Q column (1.6 $\times$ 4 cm, Amersham Phamacia Co.), 7$0^{\circ}C$ heat and acid treatment, and gel filtration column chromatography using G4000SW column (0.75 $\times$ 60cm, TOSOH Co.) by FPLC system. (omitted)

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.12
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• pp.1780-1785
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• 2005

Very low density lipoprotein (VLDL) of commercial broiler (CB) and crossbred village chicken (AK) was purified using Fast Protein Liquid Chromatography (FPLC). The fraction collected was then confirmed as VLDL using 4% polyacrylamide gel electrophoresis and transmission electron microscopy (TEM). The particle size of VLDL is 46.8${\pm}$8.6 nm. The VLDL fraction was then subfractionated and the apolipoprotein (apo) profile was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE). The CB and AK have almost similar types of apo in both subfractions 1 and 2. The AK showed the presence of apoAI, AIV, D and E whereas the CB had apo AIV, D, E and H. The apo AIV and apo E were present in both subfractions of AK and CB.

• Fisheries and Aquatic Sciences
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• v.7 no.3
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• pp.99-108
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• 2004

Vitellogenin (VTG) was purified from serum of $estradiol-l7{\beta}-treated rockfish$(Sebastes schlegeli) by precipitation with $EDTA-Mg^{2+}$ and ammonium sulfate and two step chromatography (anion exchange chromatography and gel permeation chromatography) was performed on FPLC system. Rockfish VTG (rfVTG) was characterized and its properties were determined. The monomers have apparent, molecular mass of about 188 kDa as indicated by SDS-PAGE. Amino acid composition analysis of rfVTG was similar to VTG from other oviparous teleosts. Cysteine and lysine were present at relatively high level. Leucine was present at relatively lower level than in other species. The N-terminal amino acid sequence was evaluated to identify rfVTG. Western blot analysis using an antibody against the purified VTG showed that the antibody reacted with both plasma of $estradiol-l7{\beta}-treated rockfish$ treated male and purified VTG, whereas there was no reaction with male serum of the control. An ELISA was developed using monoclonal and polyclonal antibodies against rfVTG. The assay range was 3.2 ng/mL and 1,000 ng/mL and the value of the intra and inter assay variations were within $9.7{\%}$ and $11.2{\%}$, respectively. Recovery rate was $96.8{\%}$. The sandwich ELISA could be useful for the detection of VTG and could be good for screening of estrogenic compounds.