• Korean Journal of Food Science and Technology
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• v.29 no.1
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• pp.1-8
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• 1997

A complement system-activating (anti-complementary) polysaccharide was purified from the hot water extract of young stems of Cinnamomum cassia Blume. Crude polysaccharide fraction (CC-1) was prepared from the hot water extract of the young stems followed by methanol-reflux, precipitation with ethanol, dialysis, and lyophilization. The anti-complementary activity of CC-1 was decreased greatly by periodate oxidation, but was not changed by pronase digestion. These suggest that carbohydrate moiety may be related to the activation of complement system. According to its ionic strength CC-1 was fractionated first using cetavlon to give 4 fractions, CC-2, 3, 4 and 5. Among them CC-2 fraction was found to retain the highest activity and yield. CC-2 was separated to an unabsorbed neutral sugar portion (CC-2-I) and seven absorbed acidic sugar fractions $(CC-2-II{\rightarrow}CC-2-VIII)$ on DEAE-Toyopearl 650C (Cl-). CC-2-III showing higher anti-complementary activity and yield than those of other fractions, was further purified on the gel permeation of Sephadex G-100 and Sepharose CL-6B to CC-2-IIIa-3. CC-2-IIIa-3 was determined to have a homogeneity hy GPC (Sepharose CL-6B) and HPLC. Gel chromatography using standard dextrans gave a value of $2.4{\times}10^5$ for the molecular weight. The purified polysaccharide, CC-2-IIIa-3 consisted of arabinose, xylose, glucose, galactose, galacturonic acid and glucuronic acid in a molar ratio of 5.56 : 3.77 : 1.87 : 1.00 : 5.12 : 3.13 and contained no nitrogen.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.145-145
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• 1993

일반적으로 단백질의 정제를 위한 가장 효과적인 방법은 affinity chromatography법으로 알려져 있다. 본 연구에서는 베타 수용체의 순수정제를 위한 효과적인 affinity matrix를 제조하고 이의 특징을 확인함으로써 수용체 정제를 위한 기초를 수립하고자 하였다. Bisoxirane reagent인 1,4-butanediol diglycydyl ether를 Sepharose CL-4B gel과 반응시켜 탄노원자 10개 길이의 spacer arm을 부착시켰다. 여기에 sodium persulfate, dithiothreitol을 연속적으로 사용하여 -SH기를 도입시긴 추 free radical 반응을 이용하여 베타수용체 길항제인 alprenolol을 부착시킴으로써 affinity gel을 제조하였다. alprenolol치 부착반응을 위한 최적온도는 4$0^{\circ}C$로 나타났으며, semimicro Kjeldahl 질소 분석법을 적용하여 gel을 분석한 결과 ml당 0.4-0.6 $\mu$ mol의 alprenolol이 결합하였음을 확인하였다.

• Journal of Pharmaceutical Investigation
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• v.35 no.5
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• pp.337-341
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• 2005

The purpose of the study was to prepare the pegylated liposome carrying plasmid DNA with optimal encapsulation efficiency. Plasmid DNA (pCEP4 clone 790, 10.6 kb) was entrapped in the pegylated liposome composed of neutral lipid, POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), cationic lipid, DDAB (dimethyl dioctadecyl ammonium bromide) and anionic lipids, DSPE-PEG 2000 (distearoyl phosphatidyl ethanolamine polyethylene glycol 2000) and DSPE-PEG 2000-maleimide by freezing/thawing method. Free plasmid DNA was separated from the encapsulated one by Sepharose CL-4B column chromatography. The DNA amount encapsulated into the pegylated liposome was increased as cationic lipid concentration, initial amount of plasmid DNA and total lipid amount were increased.

• Microbiology and Biotechnology Letters
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• v.23 no.5
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• pp.506-512
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• 1995

A cell aggregation factor produced by Aspergillus sp. LAM 94-142 was purified and partially characterized. The factor was purified about 15 folds from culture broth by IRA 420 and IRC 120 treatment, 1% NaCl added acetone precipitation, and Sepharose 4B column chromatography with overall yield of 48%. It was heteropolysaccharide consisted of mannose, arabinose, and glucose with a molar ratio, 31:17:2, and its molecular weight was estimated to be about 900,000 daltons by Sepharodse 4B gel filtration method. The optimum pH and temperature was 8 and 40$\circ$C, respectively. The factor was stable in pH range of 3-9 and at 100$\circ$C for 90 min. The cell aggregation activity of the factor was inhibited by the addition of Hg$^{2+}$, Fe$^{2+}$, Cu$^{2+}$, and some polypeptides such as milk casein or hemoglobin. The factor aggregated Bacillus subtilis, B. macerans, B. turingiensis, E. coli, Peudomonas aeruginosa, P. fluorescens, P. malophilia, and weakly aggregated Staphylococcus sp., Sarcina lutea, P. putida and Cryptococcus neoformnans, but it didn't aggregate various strains of Candida sp. and Saccharomyces sp.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.108-108
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• 2002

포유동물의 암컷 생식기관에 존재하는 다양한 종류의 matrix metallo-proteinase (MMP)는 난소와 자궁의 구성성분의 주기적인 변화를 조절하며 이중 난소의 MMP는 난포의 성장과 배란 그리고 퇴화 동안 조직재구성에 매우 중요한 역할을 한다고 알려져 있다. 본 실험에서는 근래에 새로 발견된 사람의 난포액에 존재하는 분자량 약 110kDa인 MMP-2 isoform GA110을 동정하고 자 하였다. 난포액으로부터 GA110 단백질을 분리하기 위하여 난포액에 5mM ethylenediaminetetraacetic acid(EDTA)를 처리한 후 DEAE Sepharose Fast Flow를 이용한 chromatography를 시행하였다. 그 결과, 난포액 단백질들은 0.2M NaCl 의 분획에서 GA110 활성을 나타내었고 anti-human MMP-2 antibody에 대한 면역반응도 뚜렷이 나타났다. DEAE Sepharose Fast Flow에서 얻은 분획 중 GA110의 활성과 면역반응을 모두 나타내는 분획만을 모아 Gelatin Sepharose 4B affinity chromatography로 다시 분리하였다 분리한 결과 resin에 흡착된 단백질 (eluate) 분획들에서 매우 뚜렷한 GA110 gelatinase 활성을 나타내었으며 면역반응 또한 관찰되었다. 이 분획들의 단백질을 농축한 후 zymography를 시행하여 나타난 GA110 단백질 band를 잘라 내었으며 이로부터 단백질을 electroelution하여 농축한 후 reducing agent인 2-mercaptoethanol를 처리하였다. 이를 전기영동 후 MMP-2 (propeptide region) antibody를 사용하여 immunoblotting 한 결과 70-72kDa의 단백질만이 면역반응을 나타내었다. 마지막으로 위와 같이 준비된 70-72kDa 단백질의 아미노산 서열을 Edman degradation 방법으로 분석하였다. 그 결과 이 단백질의 N 말단의 10개의 아미노산 배열 순서가 알려진 사람의 proMMP-2의 전체 배열순서 중 propetide domain의 N 말단에서부터 다섯 번째에서 시작하여 10개의 아미노산의 서열과 정확하게 일치하였다. 위 결과들로 미루어 사람의 난포액에 존재하는 MMP-2의 새로운 isoform인 GA110은 70-72kDa의 ProMMP-2가 disulfide bond를 통해 homodimer 구조를 이루고 있는 것으로 여겨진다.

• Archives of Pharmacal Research
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• v.19 no.4
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• pp.326-334
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• 1996

We investigated the optimum conditions for conversion of water-insoluble components of basidiocarps of Ganoderma lucidum to water-soluble components by hydrolyzing with chitinase. We also tried it with Ganoderma luciclum residue remaining after extracting hot water-soluble components of Ganoderma lucidum. After hydrolyzing under optimum conditions (20 ppm chitinase, 2% Ganoderma lucidum or 6% Ganoderma lucidum residue, at pH 3 and at $ 35^{\circ}C$), the contents of total water-soluble components (polysaccharide or protein) were measured, and it was found that the contents of water-soluble components increased to 1.5-2.7 fold. Michaelis constant, $K_m$ and maximum rate, $V_max$ calculated by Lineweaver-Burk plot for hydrolysis of Ganoderma lucidum were 1.75% and 0.02%/min respectively and those for hydrolysis of Ganoderma lucidum residue were 53.15% and 0.53%/min respectively The protein-bound polysaccharide was isolated after hydrolysis and molecular weights were measured by Sepharose CL-4B gel filtration and compared with the molecular weights of polysaccharide before hydrolysis.

• BMB Reports
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• v.28 no.4
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• pp.306-310
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• 1995

A Mono Q HR 5/5 anion-exchange column with a FPLC system was used to separate exogenously expressed calmodulin from endogenous tobacco calmodulins. Transgenic tobacco calmodulins were purified by fractionation with ammonium sulfate, precipitation with sulfuric acid and hydrophobic chromatography on phenyl-Sepharose CL-4B. The purified calmodulins were chromatographed in the FPLC using the column. This method was selected because of the slight differences in the net charge of foreign and endogenous plant calmodulins due to amino acid sequence differences. By this approach, the exogenously expressed calmodulin was isolated from endogenous tobacco calmodulins. The isolated calmodulin was characterized by amino acid composition analysis as well as methylation analysis.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.402-406
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• 1996

Inulin fructotransferase (depolymerizing) (EC 2.4.1.93) was purified 34-fold from the culture broth of Arthrobacter sp. A-6 by using a combination of ammonium sulfate fractionation, DEAE-Sepharose CL-6B chromatography and Sephacryl S-200 gel filtration. The purified enzyme converts inulin into di-D-fructofuranose dianhydride III(DFA III) and small quantities of fructo-oligosaccharides. The temperature and pH optima of the enzyme were $70^{\circ}C$ and 6.0, respectively. Molecular weight of the enzyme was determined to be 49 kDa by 12$%$ SDS-polyacrylamide gel electrophoresis, and 145 kDa by Sephacryl S-200gel filtration. This indicates that the functional inulin fructotransferase of Arthrobacter sp. A-6 has a homomeric trimer structure. The enzyme had an isoelectric point of pH 4.6. The N-terminal amino acid sequence of the purified enzyme subunit was Ala-Asp-Asn-Pro-Asp-Gly(\ulcorner)-Ser-Asn-Met(or Glu)-Tyr-Asp-Val.

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.232-239
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• 1991

Caboxymethyl cellulase (CMCase) I was purified from the induced culture filtrate of Penicllium verruculosum F-3 by ammonium sulfate precipitation, DEAE-Sephadex A-50 chromatography and Bio-gel P-150 filtration. The purified enzyme was assumed to be a glycoprotein consisting of 8.5% carbohydrate and having a molecular weight of 70.000 in SDS-polycrylamide gel electrophoresis (SDS-PAGE). The purified enzyme-specific anti-CMCase I IgG was obtained by rabbit immunization and protein A-sepharose CL-4B chromatography. The fungal poly($A^+$) RNA was isolated from the total RNA of the mycelium grown under cellulase induction conditions by oligo(dT)-cellulosse chromatography. The translation products in vitro were prepared by translating the isolated poly ($A^+$) RNA in rabbit reticulocyte lysate and analyzed by SDS-PAGE and fluorography. Of the translation products, CMCase I was identified by the immunoprecipitation against anti-CMCase I IgG.

• Food Science and Biotechnology
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• v.15 no.2
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• pp.177-182
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• 2006

Polyphenol oxidase (PPO) was isolated from the flesh of Fuji apples by DEAE-Cellulose, ammonium sulfate precipitation, phenyl-Sepharose CL-4B, and Sephdex G-100 chromatography. The molecular mass of the purified PPO was estimated to be 40 kDa by SDS polyacrylamide gel electrophoresis. With regard to substrate specificity, maximum activity was achieved with chlorogenic acid as substrate, followed by catechin and catechol whereas, there was no detectable activity with hydroquinic acid, resorcinol, or tyrosine as substrate. The optimum pH and temperature with catechol as substrate were 6.5 and $35^{\circ}C$, respectively. The enzyme was most stable at pH 6.0 and unstable at acidic pH. The enzyme was stable when it was heated to $45^{\circ}C$ but heating at $50^{\circ}C$ for more than 30 min caused 50% loss of activity. Reduced $ZnSO_4$, L-cystein, epigallocatechin-3-o-gallate (EGCG), and gallocatechin gallate (GCG) also inhibited activity.