• Journal of Microbiology
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• v.34 no.3
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• pp.255-262
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• 1996

The lipopolysaccharide (LPS) yields were measured in Rhodobacter capsulatus under several conditions by the ELISA method. The purification of LPS was done by affinity chromatography of IgG coupled CNBr-activated sepharose-4B instead of ultra-centrifugation. The purity of the LPS didn't show much difference between affinity chromatography and ultra-centrifugation method, but affinity chromatography method required much fewer organisms and was more convenient. LPS yield was measured in ng units by the ELISA method. Mannitol was a better single carbon source than other sugars, but mixing two carbon sources resulted in greater LPS yields than any sugar alone. LPS yield was directly proportional to $NH_ 4CI$ concentration, with optimum yields at 0.05% nitrogen. In contrest to LPS yields, which decreased at 0.005% nitrogen concentration total protein was increased 16 times. Calcium influenced LPS yields. At 0.7 mM $CaCI_ 2$, the LPS yield was 16.5 $\mu$g/mg DW, five times the yield without calcium.

• Bulletin of the Korean Chemical Society
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• v.33 no.11
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• pp.3665-3670
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• 2012

Metal-p-$NH_2$-Bn-DOTA (paraammionobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid: ABDOTA) complex was synthesized and purified for bio-tagging to quantify biological target materials using laser ablation (LA)-ICP-MS. Since the preparation of a pure and stable tagging complex is the key procedure for quantification, magnetic particles were used to purify the synthesized metal-ABDOTA complex. The magnetic particles immobilized with the complex attracted to a permanent magnet, resulting in fast separation from free un-reacted metal ions in solution. Gd ions formed the metal-complex with a higher yield of 64.3% (${\pm}3.9%$ relative standard deviation (RSD)) than Y ions, 52.3% (${\pm}2.5%$ RSD), in the pH range 4-7. The complex bound to the magnetic particles was released by treatment with a strong base, of which the recovery was 81.7%. As a reference, a solid phase extraction (SPE) column packed with Chelex-100 resin was employed for separation under similar conditions and produced comparable results. The tagging technique complemented polydimethylsiloxane (PDMS) microarray chip sampling in LA-ICP-MS, allowing determination of small sample volumes at high throughputs. For application, immunoglobulin G (IgG) was immobilized on the pillars of PDMS microarray chips and then tagged with the prepared Gd complex. IgG could then be determined through measurement of Gd by LA-ICP-MS. A detection limit of 1.61 ng/mL (${\pm}0.75%$ RSD) for Gd was obtained.

• The Plant Pathology Journal
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• v.31 no.4
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• pp.379-387
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• 2015

Melon necrotic spot virus (MNSV) was recently identified on watermelon (Citrullus vulgaris) in Korea, displaying as large necrotic spots and vein necrosis on the leaves and stems. The average occurrence of MNSV on watermelon was found to be 30-65% in Hapcheon and Andong City, respectively. Four isolates of the virus (MNSV-HW, MNSV-AW, MNSV-YW, and MNSV-SW) obtained from watermelon plants in different areas were non-pathogenic on ten general indicator plants, including Chenopodium quinoa, while they infected systemically six varieties of Cucurbitaceae. The virus particles purified by 10-40% sucrose density gradient centrifugation had a typical ultraviolet spectrum, with a minimum at 245 nm and a maximum at 260 nm. The morphology of the virus was spherical with a diameter of 28-30 nm. Virus particles were observed scattered throughout the cytoplasm of watermelon cells, but no crystals were detected. An ELISA was conducted using antiserum against MNSV-HW; the optimum concentrations of IgG and conjugated IgG for the assay were $1{\mu}l/ml$ and a 1:8,000-1:10,000 dilutions, respectively. Antiserum against MNSV-HW could capture specifically both MNSV-MN from melon and MNSV-HW from watermelon by IC/RT-PCR, and they were effectively detected with the same specific primer to produce product of 1,172 bp. The dsRNA of MNSV-HW had the same profile (4.5, 1.8, and 1.6 kb) as that of MNSV-MN from melon. The nucleotide sequence of the coat protein of MNSV-HW gave a different phylogenetic tree, having 17.2% difference in nucleotide sequence compared with MNSV isolates from melon.

• BMB Reports
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• v.34 no.6
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• pp.509-516
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• 2001

A genomic DNA fragment that contains the gene, which codes for a novel extracellular serine protease in Burkholderia pseudomallei, was cloned by using pQE40 as a vector. It was maintained in Escherichia coli JM109. The expression of the gene(s) resulted in the production of a 52 kDa protease. The recombinant protease was purified from the culture filtrate via ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography. The purified protease had an optimum pH and temperature of pH 8.9 and $38^{\circ}C$, respectively. The protease activity was inhibited by EGTA, EDTA, and PMSF, but not 1,10-phenanthroline. The first 11 amino acid residues from the N-terminus of the purified protease were identified as LAPNDPYYYGY. PNDPYY was found to show homology to the Bacillus cereus microbial serine protease and B. subtilis PD498 serine protease. These results indicate that the protease that was purified in this study is an extracellular calcium-dependent serine protease. The purified protease was able to digest the human serum 19A, IgG, albumin, and transferrin, as well as bovine muscle actin and myosin. Furthermore, it was able to promote or cause dermonecrosis in experimental rabbits. These results propose the possible role of a novel B. pseudomallei extracellular calcium-dependent serine protease in the virulence of the pathogen.

• Microbiology and Biotechnology Letters
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• v.17 no.1
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• pp.19-23
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• 1989

A sequential system composed of hydroxylapatite chromatography and gel permeation chromatography was developed to purify the IgM type monoclonal antibody against the colon cancer cell SC-1 from the ascitic fluid of mice injected with the murine hybridoma CH07E02. In the hydroxylapatite chromatographic step the band dilution could be reduced by controlling the gradient and flow rate of the eluent, the sodium phospate buffer, the optimum values for these variables being 5.82$\times$10$^{-3}$M/cm and 0.2$m\ell/\textrm{cm}^2$/min, respectively. A degree of purity better than 99.99% as judged from silverstaining of the SDS-PAGE bands, was obtained by adding the gel permeation chromatographic step in tandem.

• Microbiology and Biotechnology Letters
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• v.20 no.2
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• pp.225-232
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• 1992

In order to develop an enzyme-linked immunosorbent assay(ELISA) for detecting aflatoxin $B_1(AFB_1)$, we produced and purified antibodies, thereafter established and evaluated methods of direct and indirect competitive ELISA. Anti-AFB, antisera, produced by immunizing rabbits with $AFB_1$-1-(0-carboxymethy1)oxime-bovine serum albumin conjugate ($AFB_1$-BSA), were removed of anti-BSA antibodies by quantitative precipitation reaction and further purified by ammonium sulfate precipitation and DEAE-Sephadex A-50 ion exchange chromatography. Purified IgG fractions were used as anti-$AFB_1$ antibodies. The antibodies, whose titer was deterrnined extremely high above $2 \times 10^6$, showed low cross-reactivity of 3~34% against $AFB_1$ analogues such as G2, B2, and GI. From the standard curves of direct and indirect competitive ELISA for AFBI, the detection ranges were found 0.2~20 and 1~10, 000 ng/ml(ppb) respectively. In their sensitivity, stability, simplicity, and rapidity, the direct method was more suitable than the indirect method for practical use.

• BMB Reports
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• v.47 no.3
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• pp.130-134
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• 2014

The combination of a high-affinity antibody to a hapten, and hapten-conjugated compounds, can provide an alternative to the direct chemical cross-linking of the antibody and compounds. An optimal hapten for in vitro use is one that is absent in biological systems. For in vivo applications, additional characteristics such as pharmacological safety and physiological inertness would be beneficial. Additionally, methods for cross-linking the hapten to various chemical compounds should be available. Cotinine, a major metabolite of nicotine, is considered advantageous in these aspects. A high-affinity anti-cotinine recombinant antibody has recently become available, and can be converted into various formats, including a bispecific antibody. The bispecific anti-cotinine antibody was successfully applied to immunoblot, enzyme immunoassay, immunoaffinity purification, and pre-targeted in vivo radioimmunoimaging. The anti-cotinine IgG molecule could be complexed with aptamers to form a novel affinity unit, and extended the in vivo half-life of aptamers, opening up the possibility of applying the same strategy to therapeutic peptides and chemical compounds.

• Parasites, Hosts and Diseases
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• v.52 no.1
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• pp.117-120
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• 2014

Several studies have reported that the citrus red mites Panonychus citri were an important allergen of citrus-cultivating farmers in Jeju Island. The aim of the present study was to purify and assess properties of a cysteine protease from the mites acting as a potentially pathogenic factor to citrus-cultivating farmers. A cysteine protease was purified using column chromatography of Mono Q anion exchanger and Superdex 200 HR gel filtration. It was estimated to be 46 kDa by gel filtration column chromatography and consisted of 2 polypeptides, at least. Cysteine protease inhibitors, such as trans poxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) and iodoacetic acid (IAA) totally inhibited the enzyme activities, whereas serine or metalloprotease inhibitors did not affect the activities. In addition, the purified enzyme degraded human IgG, collagen, and fibronectin, but not egg albumin. From these results, the cysteine protease of the mites might be involved in the pathogenesis such as tissue destruction and penetration instead of nutrient digestion.

• Journal of the Korean Magnetic Resonance Society
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• v.5 no.1
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• pp.46-55
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• 2001

P23, a translationally controlled turner protein is involved in the interleukin-4 secretion from human basophils and is also known to be an IgE-dependent histamine-releasing factor. However, the precise physiological function and structure of P23 have not been elucidated. In the current study, we constructed the optimal expression and purification protocol of P23 and investigated the secondary structure and structural stability in various conditions. Circular dichroism (CD) investigation showed that the secondary structure of P23 adopts mainly a P-sheet conformation. CD spectroscopy and differential scanning calorimetry revealed that P23 is fairly stable in the pH range of neutral and mild-basic conditions and in the temperature range of 10 - 50$\^{C}$. Since the thermal stability and the P-sheet content of P23 were decreased by the addition of Ca$\^$2+/ ion, it could be suggested that Ca$\^$2+/ion induces structural change by partially destabilizing the structure of P23. In addition various H experiments were monitored to solve the aggregation of P23. Den results will provide the preliminary structural information about P23.

• YAKHAK HOEJI
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• v.42 no.4
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• pp.395-402
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• 1998

Mori Cortex Radicis, the root bark of mulberry tree, has been used in the treatment of bronchial asthma and other lung diseases in traditional medicine. There was a recent repor t that the water soluble part with molecular weight of above 10,000 has anti-allergic activity. Therefore, we intended to isolate and purify the anti-allergic compound from hot water extract of the Mori Cortex Radicis. Crude extract of Mori Cortex Radicis was prepared by hot-water extraction, and anti-allergic compound was further purified by alcohol precipitation, successive ultrafiltration, anion exchange chromatography and gel filtration chromatography. This compound had homogeneity which was shown by the sharp single peak in HPLC chromatogram (TSK-GEL G400OPW column, RI detector). The molecular weight of the compound was estimated as 23Kda on the basis of calibration curve plotted against protein standards. This compound was identified as complex of sugar, protein and lignin (19.2: 5.9: 72.7), and proteolysis could not decrease the anti-allergic activity but mild delignification decreased the activity remarkably. Therefore, we concluded that the anti-allergic compound of Mori Cortex Radicis was a lignin-carbohydrate complex.