• The Journal of Natural Sciences
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• v.14 no.2
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• pp.67-82
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• 2004

A thiol-specific antioxidant protein (TSA or TPx) was purified from Halophilic archeabacteria Halococcus agglomeratus, by DEAE-Cellulose, Phnyl, sepharose, Sephadex G-75, Sephacryl S-100, Sephacryl S-200, and Q-Wepharose FF. This protein exhibited the preventeive effect against the inactivation of glutamine synthehase (GS) activity was support by a thiol-reducing equicalent such as dithiothreitol. TPx activity was maximal at NaCl concentration above 500mM. The molecular mass of the protein was determinated to be 22-kDa by SDS-PAGE. The TPx purified from Halococcus agglomeratus seems to be similar to other TPx family, except for the salt requirement for the maximal antioxidant activity.

• Journal of Microbiology
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• v.33 no.3
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• pp.244-251
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• 1995

An extracellular protease of Salmonella schottmulleri was purified from culture filtrate by using 0-75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, Ultrogel HA chromatography and Sephacryl S-200 HR molecular sieve chromatography. To measure enzyme activity, synthetic dipeptide substrate (CBZ-arg-arg-AFC) with low molecular weight was employed as substrate. The molecular weight of the purified enzyme was approximately 80 kDa when determined by gel filtration on Sephacryl S-200 HR and 73 kDa when estimated by SDS-PAGE. The isoelectric point was 5.45. The activity of the purified enzyme was inhibited by metal chelating agesnts such as EDTA and 1.10-phenanthroline. The divalent cations, such as Ca$\^$2+/, Zn$\^$2+/, Fe$\^$2+/, Mg$\^$2+/ enhanced its activity. These results suggested that it was a metalloprotease. It had a narrow pH optimum of 6.5-7.5 with a maximum at pH 7.0 and a temperature optimum of 40.deg.C. It was stable at least for 1 week at 40.deg.C and maintained its activity for 24 hours at 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium dodecyl sulfate (SDS) and was inactivated in a dose-dependent manner. However, it was resistant to Triton X-100 and the activity was enhanced to 32.3% with treatment of 0.025% Triton X-100.

• Journal of Life Science
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• v.13 no.3
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• pp.308-313
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• 2003

The collagenase gene from Vibrio parahaemolyticus 04 was subcloned into an expression vector pET-29b. The recombinant collagenase was expressed in Escherichia coli BL2l(DE3) and partially purified by Hi-Trap affinity and Sephacryl S-100 size exclusion chromatographies. The recombinant enzyme was purified by 43.7-fold and the yield was 73%. SDS-PAGE revealed that the molecular weight of the enzyme was approximately 35 kDa. Substrate specificity study of the enzyme displayed that the enzyme showed the highest activity with the type I collagen and the synthetic peptide, Z-GPGGPA, indicating that the enzyme was indeed a collagenase. The enzyme showed broad pH optimum around pH 6-12 and was stable between pH 5.5 and 11.5. The optimum temperature for the type I collagen degradation was $35^{\circ}C$. The thermostability measurement of the enzyme indicated that the enzyme was stable up to $55^{\circ}C$, but the activity was diminished quickly above $60^{\circ}C.$

• Food Engineering Progress
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• v.13 no.4
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• pp.326-334
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• 2009

Blood coagulation and aggregation of platelet are crucial events in the pathogenesis of various ischemic diseases. The substance which can prevent blood coagulation and platelet aggregation was extracted from wood ear mushroom (Auricularia auricular-judae) and its anticoagulation activity was investigated. The dried A. auricular-judae was extracted with 0.1 N NaOH and its supernatant was further extracted with methanol and ethanol followed by $H_{2}O$. The resulting methanol soluble fraction showed significant antithrombotic activity in activated partial thromboplastin time, thrombin time, and prothrombin time assays with values of 100, 124, and 54 sec, respectively. This active substance was purified with DEAE-Sepharose CL6B and Sephacryl 400-HR and was found to be polysaccharide with the average molecular weight of over 150 kDa. This polysaccharide was xyloglucomannan of which the main component was mannose, and its anticoagulant activity was mostly mediated by inhibition of thrombin activity.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.180-180
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• 1994

Farnesyl protein transferase는 Ras precursor의 C-terminal에 있는 cystein residue에 farnesyl group을 결합시키는 효소다. 이 효소를 bovine testis에서 30-50% ammonium sulfate fractionation, DEAE-sephacel ion exchange, Sephacryl s-300 gel filtration, hexapeptide(KKCVIM) affinity chromatography를 통해 30000배로 분리하였다. 분리된 효소는 gel filtration시 약 100kDa으로, SDS-polyacrylamide 전기영동시 50kDa의 인접한 두 bands로 나타났고 이것은 $\alpha$, $\beta$ subunits으로 생각되었다. $\alpha$ subunit을 encoding하는 RAM2 유전자를 site directed mutagenesis로 145번의 histidine을 aspartate로, 140번의 aspartate를 asparagine 으로 바꾸었더니 optimal pH와 $K_{m}$ 값이 변했다. Diethyl pyrocarbonate로 histidine residues를 chemical modification시켰을때 효소의 활성이 저하되었다. 145번 histidine이 aspartate로 바뀐 돌연변이효소에서 비교적 느리게 활성이 저하되므로 145번 histidine이 이 효소의 active site에 있을것으로 추측된다.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.55-55
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• 1993

Farnesyl Protein transferase(FPT)는 발암유전자 ras의 단백질 산물인 p$^{21}$의 post-translational modification의 첫 단계인 ras-farnesylation에 관여하는 효소로 본 연구에서는 정제된 FPT와 E. coli에서의 발현 system을 이용하여 FPT의 구조와 기능을 밝히고 이를 FPT 방해제의 설계에 이용하고자 한다. Bovine testis에 존재하는 FPT를 30%-50%의 Ammonium sulfate로 fractionation하고, DEAE-Sephacel, Sephacryl S-300 column을 통과시킨 후 peptide(KKCVIM) affinity column을 이용하여 순수 정제하였다. 정제된 효소의 분자량은 gel-filtration에 의해 100KDa으로 추정되었고 SDS-PAGE 결과 49KDa과 46KDa의 두 subunit로 구성되었음이 확인되었다. 효소활성에는 $Mg^{2＋}$ 와 $Zn^{2＋}$가 필수적이며 최적 pH는 7.0이었다. Yeast의 FPT의 두 subunit 유전자는 Yeast genomic DNA를 template로 사용하고 각 subunit에 specific한 합성된 primer들과 vent polymerase를 이용하여 Polymerase chain reaction을 통하여 얻었다. 두 유전자를 pBluescriptII SK＋ vector를 변형시킨 두 vector, pBSK＋4와 pBChl＋4에 재조합 시킨 후 E.coli에 transformation시켜 발현시켰다. 현재 정제된 Bovine FPT와 E. coli에서 발현된 Yeast FPT의 chemical modification과 site-directed mutagenesis를 통하여 FPT의 active site와 substrate binding site에 관한 연구를 진행시키고 있다.

• Korean Journal of Food Science and Technology
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• v.20 no.6
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• pp.780-786
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• 1988

In order to investigate the most efficient and rapid method for the purification of enterotoxin A from Staphylococcus aureus M 7/1, various methods such as ion-exchange chromatography on Amberlite, and CM-cellulose. gel filtration on Sephadex G-50, 75, 100 and Sephacryl, and fast protein liquid chromatography (FPLC) were applied and compared in terms of purity and speed. Although ion-exchange chromatography on Amberlite resin was good enough to remove other materials in culture medium from enterotoxin, and convenient, and fast method, the purity of this method was less than 70%. However. carboxymethyl ion-exchange column showed to be better purity than that of Amberlite method. The yields of these two methods were about 70% and 75%, respectively. When gel filtration methods on Sephadex G-50, 75, 100 and Sephacryl were applied, the purity was about 90%. Fast protein liquid chromatography was found to be the most efficient method in terms of purity (97%) and speed. The combined method, gel filtration after CM-cellulose column (stepwise elution) treatment can be also used as a efficient method particularly for the purification of large volume of sample.

• Korean Journal of Veterinary Research
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• v.38 no.2
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• pp.328-335
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• 1998

Outer membrane proteins(OMPs) of Brucella abortus 1119-3 strain were extracted by Triton X-100 treatment, and fractionated by DEAE-cellulose column chromatography and Sephacryl S-300 column chromatography. The antigenic proteins in these fractions were identified by Western blot analysis. In Western blot analysis, a single band(38kDa) was observed in the DEAE fractions from 36th fraction to 38th fraction against sera of cattle infected with B abortus. And other fractions have several bands. However, the Sephacryl S-300 fractions exhibited a total of 3 peaks of proteins with a broad range from about 30 to 116kDa. In order to characterize further, the extracted OMPs and the DEAE fractions were analyzed by two dimensional polyacrylamide gel electrophoresis(2-DE) and Western blot using serum from naturally infected cattle with Brucella spp. The 2-DE immunoblots of DEAE fraction showed immunoreactive spots more than twenty two. The major protein spots have ranging from about 32 to 47kDa. The pI values of the spots were detected from pH 4.7 to 5.4. Among the major protein spots, the 38kDa protein which is a specific antigen, located at the point of approximately a pI 4.8.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.126-126
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• 1995

본 연구는 중금속에 의한 생체내 독성기전을 연구하고자 흰쥐간의 상층액에서 카드뮴과 잘 결합하는 HMWP들을 분리, 정재하고 나아가 생화학적 특성을 밝히고 이단백질이 카드뮴에 의해 생성되는 것인지 아니면 간의 기존 단백질인지를 밝히고자 함을 연구의 목적으로 하였다. CdCl %2 (3mg/kg body wt.)을 3일간 ip injection시킨 후 흰쥐의 간을 적출하고 균질화하여 원심분리한 crude extract를 직접 Sephacryl S-100에 흡착시켜 10mM phosphate buffer(pH 7.0)으로 용출시켰다. 용출된 fraction tube를 UV spectrometer로 흡광도를 측정하고 atomic absorption spectrophotometer로 카드뮴량을 측정하여 카드뮴량이 높은 분획을 모아서 ion exchange column chromatography(DEAE-Sepharose)에 흡착시켜 염농도 구배로 chromatography를 실시하고, 음이온 교환수지에서 흡착되지 않고 용출된 분획을 ultrafiltration으로 농축시킨 후 S-Sepharose에 흡착시켜 염농도 구배로 chromatography를 실시한 결과 두 종류의 카드뮴 결합 단백질(Cd-BP)를 분리, 정제하였다.

• Journal of Life Science
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• v.15 no.4 s.71
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• pp.657-663
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• 2005

Collagenases are generally defined as enzymes that are capable of degrading the polypeptide backbone of native collagen under conditions that do not denature the protein. An extracellular collagenase-producing bacterial strain was isolated from kimchi and identified to be Bacillus subtilis JS-17 through morphological, cultural, biochemical characteristics and 16S rDNA sequence analysis. Optimum culture condition of Bacillus subtilis JS-17 for the production of collagenase was $1.5\%$ fructose, $1\%$ yeast extract, $0.5\%\;K_2HPO_4,\;0.4\%\;KH_2PO_4,\;0.01\%\;MgSO_4\cdot7H_2O,\;0.01\%\; MnSO_4\cdot4H_2O,\;,0.1\%$ citrate and $0.1\%\;CaCl_2$. The production of collagenase was optimal at $30^{\circ}C$ for 72 hr. A collagenase was isolated from the culture filtrate of Bacillus subtilis JS-17. The enzyme was purified using Amberlite IRA-900 column chromatography, Sephacryl S-300 HR column chromatography and DEAE-Sephadex A-50 column chromatography The purified collagenase has an specific activity 192.1 units/mg. The molecular weight of the purified enzyme was estimated to be 28 kDa by SDS-PACE. The purified collagenase has $100\%$ activity up to $55^{\circ}C$.