• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.539-542
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• 2003

SDS-PAGE 결과 elution pH에 따라 분리되는 band pattern은 비슷하게 2band의 양상을 보이지만, FI값을 비교하여 보았을 때 다른 pH보다 8.0에서 가장 높은 수준을 보였으므로 GFPuv/cytochrome c-552 fusion Protein의 분리 ${\cdot}$ 정제에 가장 적합한 pH를 8.0으로 정하였다.

• 한국동물학회지
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• 제29권3호
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• pp.190-200
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• 1986

논우렁이의 組織別 蛋白質 패턴에 있어서 筋肉은 雌雄에 關係없이 16個의 主要蛋白質 bands는 거의 同一하였으며, 그 중에서 bands E (Mr. 41,500), H (Mr. 52,100), L (Mr. 71,700), N(Mr. 98,500), O (Mr. 107,900) 와 P (Mr. 112,900)는 筋肉과 精巢 卵巢에서 나타나는 蛋白質이었다. 그러나 精巢나 卵巢의 蛋白質 패턴은 그들 固有의 蛋白質 패턴을 가지고 있었으며 組織에서 全部 나타나는 band外에 精巢와 卵巢에서 共同으로 나타나는 主要한 bands는 약 5個[bands d (Mr. 15,600), k (Mr. 37,100), p (Mr. 57,000), s (Mr. 80,300), v (Mr. 105,400)]로 나타남을 알 수 있었다. 그리고 主要蛋白質 bands를 elution 시켜서 아미노산을 分析한 結果는 단백질 패턴의 양상과 같이, 아미노산의 量과 組成이 많은 순서는 卵巢, 筋肉, 精巢의 順으로 되었다. 이러한 結果를 볼 때 卵巢에서는 全個體에서 가질수 있는 豊富한 蛋白質이 發見되는 同時에 精巢에서는 核酸物質의 濃縮으로 情蟲이 되기 위한 morphogenesis의 現象으로 말미 암아 蛋白質의 量이 매우 적게 나타나는 것으로 生覺되었다.

• Parasites, Hosts and Diseases
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• 제37권1호
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• pp.39-46
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• 1999

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection. a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HC1 (pH 7.4) containing 0.05. 0.1, 0.2 and 0.4 M NaC1 in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1. 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease. showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may playa role in the nutrient uptake of N. seoulense from the host intestine.

• BMB Reports
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• 제29권5호
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• pp.436-441
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• 1996

The palmitoyl acyl carrier protein (ACP) specific thioesterase (EC 3.1.2.14) from Iris pseudoacorus was purified and characterized. The thioesterase which was very unstable in relatively high salt concentrations was eluted using a co-gradient of Triton X-100 and low concentration of KCl or Na-phosphate from Q-Sepharose, DEAE-Sepharose, and hydroxyapatite chromatography. SDS-PAGE analysis showed a single band with a molecular weight of 35,000. The native molecular weight of approximately 37,000 was estimated by Sephacryl S-200 chromatography, indicating that the enzyme is a monomer. The thioesterase activity was inhibited about 75% and 50% by N-ethylmaleimide (2 mM) and phenylmethylsulfonyl fluoride (2 mM). respectively. The N-ethylmaleimide-inactivation was protected by sodium palmitate but the inactivation with phenylmethylsulfonyl fluoride was not protected. Oxidation of thiols by 2 mM 5.5'-dithio-bis-(2-nitrobenzoic acid) resulted in 65% inactivation of the enzyme. These results suggest that a cysteinyl residue is essential to the catalytic reaction of the enzyme. The enzyme activity was increased by sodium citrate and also by $Cu^{2+}$

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.912-918
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• 2003

Mitotic centromere-associated kinesin (MCAK), which is a novel kinesin with a central motor domain, is believed to playa role in mitotic segregation of chromosome during the M phase of the cell cycle. In the present study, it is shown that a rabbit polyclonal antibody has been produced using the N-terminal region (187 aa) of human MCAK expressed in E. coli as the antigen. To express the N-terminal region in E. coli, the MCAK cDNA fragment encoding N-terminal 187 aa was obtained by PCR and was then inserted into the pET 3d expression vector. Molecular mass of the N-terminal region overexpressed in the presence of IPTG was 23.2 kDa on SDS-PAGE, and the protein was insoluble and mainly localized in the inclusion body that could be easily purified from the other cellular proteins. The N-terminal region was purified by electro-elution from the gel after the inclusion body was resolved on the SDS-PAGE. The antiserum obtained after tertiary immunization with the purified protein specifically recognized HsMCAK when subjected to Western blot analysis, and showed a fluctuation of the protein level during the cell cycle of human Jurkat T cells. Synchronization of the cell-cycle progression required for recovery of cells at a specific stage of the cell cycle was performed by either hydroxyurea or nocadazole, and subsequent release from each blocking at 2, 4, and 7 h. Northern and Western analyses revealed that both mRNA and protein of HsMCAK reached a maximum level in the S phase and declined to a basal level in the G1 phase. These results indicate that a polyclonal antibody raised against the N-terminal region (187 aa) of HsMCAK, overexpressed in E. coli, specifically detects HsMCAK (81 kDa), and it can analyze the differential expression of HsMCAK protein during the cell cycle.

• BMB Reports
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• 제32권2호
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• pp.133-139
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• 1999

Thioltransferase, also known as glutaredoxin, was previously purified and characterized from Chinese cabbage (Brassica campestris ssp. napus var. pekinensis). However, in the process of gel filtration on Sephadex G-75, there were two activity peaks. In this study, a second thioltransferase (TTase CC-2) in the minor peak of the Sephadex G-75 elution profile was further purified using affinity chromatography on an S-hexylglutathione-agarose column by eluting with buffer solution containing 2.5 mM S-hexylglutathione. It showed a single band on SDS-PAGE indicating that TTase CC-2 is electrophoretically homogeneous. The molecular weight of TTase CC-2 was estimated to be about 22,000 daltons, and its isoelectric point was determined to be 6.73. Its size appears to be atypical and much larger than that of the first thioltransferase (TTase CC-1) from Chinese cabbage, and it can utilize 2-hydroxyethyl disulfide, S-sulfocysteine, and insulin as substrates. S-sulfocysteine was found to be a superior substrate for TTase CC-2. TTase CC-2 also displayed the reducing activity for non-disulfides such as dehydroascorbic acid. Its optimum pH was 8.5, which was consistent with that of TTase CC-1. TTase CC-2 activity was greatly activated by L-cysteine and reduced glutathione, and was found to be less heat-stable compared with TTase CC-1. Molecular and physiological differences between TTase CC-1 and TTase CC-2 remain to be elucidated. Chinese cabbage is the first plant which is known to contain two kinds of thioltransferases.

• KSBB Journal
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• 제18권2호
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• pp.122-126
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• 2003

본 연구에서는 다중 이온교환크로마토그래피로 리소짐을 분리하는 공정개발을 위해 Cellufine CM C-200과 Bio-rex 70 겔을 이용하여 비교 실험하였다. 난백에서 Iysozyme의 분리, 정제 및 수지의 활용성을 확인하는 실험이었다. 실험결과 Cellufine겔은 5 주기가 지나면서부터 용출분획에서 낮은 농도의 단백질이 용출됨을 확인하였다. 이는 주기가 진행될 수록 겔의 흡착능력이 약화됨을 보여주는 것이다. 또한 주기가 진행될수록 각 주기의 크로마토그램을 분석한 결과, 단백질이 겔의 작용기와 효과적으로 흡·탈착 작용을 하고 있지 못하기 때문에 용출영역의 peak가 계속해서 낮아지는 경향을 보였다. 그러나 Bio-rex 70겔은 6 주기를 수행 후에도 겔의 흡착능력이 상실되지 않고 단백질과 효과적으로 결합함을 용출영역의 크로마토그램과 전기영동에서 확인하였다. Bio-rex 겔은 Cellufine겔보다 단백질과 작용기와의 결합력이 우수하였으나 순수한 Iysozyme의 정제에 있어서 적당하지 못함을 알 수 있었다. Iysozyme의 용출 이온강도가 다른 단백질들보다 더 강하기 때문에 용출용액의 소금 농도구배를 실행함으로써 단백질을 용출한다면 순수한 IysoByme을 정제할 수 있다고 생각된다. 따라서 본 실험결과 다중 이온교환크로마토그래피 공정에 효과적인 겔은 Bio-rex 70겔임을 알 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제23권2호
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• pp.167-176
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• 2013

A novel bioactive molecule produced by Bacillus thuringiensis subsp. kurstaki Bn1 (Bt-Bn1), isolated from a common pest of hazelnut, Balaninus nucum L. (Coleoptera: Curculionidae), was determined, purified, and characterized in this study. The Bt-Bn1 strain was investigated for antibacterial activity with an agar spot assay and well diffusion assay against B. cereus, B. weinhenstephenensis, L. monocytogenes, P. savastanoi, P. syringae, P. lemoignei, and many other B. thuringiensis strains. The production of bioactive molecule was determined at the early logarithmic phase in the growth cycle of strain Bt-Bn1 and its production continued until the beginning of the stationary phase. The mode of action of this molecule displayed bacteriocidal or bacteriolytic effect depending on the concentration. The bioactive molecule was purified 78-fold from the bacteria supernatant with ammonium sulfate precipitation, dialysis, ultrafiltration, gel filtration chromatography, and HPLC, respectively. The molecular mass of this molecule was estimated via SDS-PAGE and confirmed by the ESI-TOFMS as 3,139 Da. The bioactive molecule was also determined to be a heat-stable, pH-stable (range 6-8), and proteinase K sensitive antibacterial peptide, similar to bacteriocins. Based on all characteristics determined in this study, the purified bacteriocin was named as thuricin Bn1 because of the similarities to the previously identified thuricin-like bacteriocin produced by the various B. thuringiensis strains. Plasmid elution studies showed that gene responsible for the production of thuricin Bn1 is located on the chromosome of Bt-Bn1. Therefore, it is a novel bacteriocin and the first recorded one produced by an insect originated bacterium. It has potential usage for the control of many different pathogenic and spoilage bacteria in the food industry, agriculture, and various other areas.

• 한국수산과학회지
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• 제27권2호
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• pp.107-113
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• 1994

우리나라 남해연안 어패류에서 V. vulnificus, V. cholerae non O1 균을 분리하여 이들 균이 생산한 용혈독소의 활성을 검토하고 특히 치사율이 높은 패혈증 원인균인 V. vulnificus균이 생산한 균체외 단백독소인 hemolysin을 분리정제하고 얻어진 독소를 이용하여 항혈청을 만들었다. 1. V. vulnificus hemolysin(VVH)은 HI broth에서 $37^{\circ}C,\;15{\sim}24hr$ 진탕배양으로 잘 생산되었으며 V. cholerae non O1 균의 경우는 배양 15시간까지는 hemolysin 생산이 증가되었으나 15시간 경과 후에는 균종에 따라 증가되는 것도 있었고 경과 시간에 따라 오히려 감소하는 균주도 있었다. 2. V. vulnificus가 생산한 hemolysin은 면양적혈구에 대한 용혈활성이 강하고 토끼적혈구에 대하여는 약하였으나 V. cholerae non O1 균주는 토끼적혈구에 대한 용혈활성이 면양이나 말 적혈구에 대한 활성보다 2배정도 강하였다. 3. VVH는 hydrophobic Phenyl-Sepharose HP column을 이용하여 washing buffer와 elution buffer의 성분과 pH를 조정하면서 $1\%$ CHAPS를 이용하여 2차에 걸쳐 column chromatography한 결과 정제도와 수율이 매우 좋아졌다. 본 방법으로 다섯 차례에 걸쳐 정제한 결과 정제된 VVH의 specific activity는 $16900{\sim}52300$배로 평균 27,000배 이상 증가하였으며 수율도 $18.2{\sim}33.0\%$로 평균 $23.4\%$나 되었다. 실제로, V. vulnificus 배양액 2400ml로 부터 정제된 hemolysin을 $250{\mu}g$정도 만들 수 있어서 패혈증 비브리오균 연구에 크게 이바지 할수 있을 것으로 사료된다. 4. 정제된 VVH를 SDS-PAGE한 결과 분자량은 50KDa이었으며 토끼를 이용해서 만든 항혈청의 항체가는 $2000{\sim}8500$이었다.

• 농촌의학ㆍ지역보건
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• 제22권1호
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• pp.61-74
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• 1997

In case of tissue invading nematode, proteolytic enzyme was required at their parasitic life. Proteinases obtained from these parasites(Toxocara canis, Ansakis spp. and Trichinella spiralis) were extracted, isolated and further purified. And then the analysis for activity and inhibitory effect of proteinases were performed by appropriate substrate. Determination of protein as a circulating antigen was done in use of infected animal serum with above parasites, respectively. For above experimental objects, following procedures were performed. First, enzymatic activity was measured in use of azocasein and inhibitory effect of porteinase were studied by various inhibitors. Second, partially purified proteins containing enzymatic activity were obtained by ion exchange chromatography, ultrafiltration and electrophoretic elution. Third, role of the partially purified protein as a circulating antigen. The results obtained were as follows : 1. Enzymatic activity of each nematode proteinase was varied according to pH. Optimal pH of Toxocara canis, Ansakis spp. and Trichinella spiralis were pH 6.0, pH 5.5 and pH 6.5, respectively. The optimal molarity of buffer was 0.1M phosphate buffer. Although little difference between these proteinases was observed, temperature stability was at least maintained at $4^{\circ}C$ until 5 days. 2. In case of Ansakis spp. and Toxocara canis, enzymatic activity of these proteinases was considerably inhibited by Leupeptin and EDTA. For maximum enzymatic activity of above proteinases, it was required that cysteine residue of enzyme should be protected. And it was suggested that metallo type was contained in enzyme active site. Proteinase of Trichinella spiralis contained metallo type also. 3. Although partial purification was performed in Ansakis spp. and Toxocara canis, proteins maintaining enzymatic activity were identified as a circulating antigen. From SDS-PAGE and immunoblot, 25 kDa was presented in Ansakis spp.. Specific antigen of Toxocara cains was 110 kDa protein fraction. 55 and 42 kDa proteins were reacted with normal serum. Trichinella spiralis 60 kDa protein fraction was successfully purified from excretory materials in culture. As a result of immune-reaction with Trichinella spiralis infected serum, highly purified 60 kDa protein was maintained antigenicity until final purification step.