• Journal of the Korea Academia-Industrial cooperation Society
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• v.22 no.6
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• pp.542-549
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• 2021

The purpose of this study was to provide an economical and easy preparation method for recombinant human epidermal growth factor (rhEGF) without the need for an expensive enzyme to cleave the fusion part. However, the N-terminal fusion part is still useful for affinity chromatography. The hEGF is an important hormone in cell growth and proliferation in humans, and many studies on the expression and purification of this protein have been reported. In the present study, the hEGF gene was designed to be optimized with the E. coli codon usage preference and to contain Asn-Gly at the N-terminus of the protein. The gene was inserted into pRSET_A, an E. coli expression vector, and transformed into E. coli BL21 (DE3). The recombinant fusion protein was successfully co-expressed with pG-Tf2, a chaperone vector, in E. coli and purified by Ni-NTA column chromatography. The rhEGF was then released by hydroxylamine treatment and confirmed by SDS-PAGE. ELISA analysis showed that the activity of the free rhEGF was more than 92% similar to that of commercial EGF. The biological activity of the rhEGF was confirmed by a cell proliferation test with human skin fibroblasts.

• Applied Biological Chemistry
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• v.38 no.5
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• pp.371-375
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• 1995

The antagonistic rhizobacteria Pseudomonas(P.) fluorescens against F. oxysporum and R. solani were isolated and selected, and then, their biological and physiological characteristics were investigated. The posibility and optimum condition of the electroporation of antagonistic rhizobacteria with Ps70, one of the selected one, and plasmid pSV2-neo was studied. Its optimum condition was found with HGEB which contains 1 mM (pH 7.0) hepes and 10% glycerol at setting of 200 resistance, 25 ${\mu}F$ capacitance, and 2.5 kV applied voltage. In addition, the transformation efficiency obtained with pSV2-neo was compared to other plasmids with different sizes. The applied voltage, the buffer composition and the parallel resistor (time constant) were shown to have the greatest effect on transformation efficiency in electroporation. And the rest of the selected rhizobacteria were also successfully transformed with pSV2-neo by electroporation.

• Journal of Korean Society of Environmental Engineers
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• v.37 no.4
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• pp.246-252
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• 2015

In steel factory, hot roller cleaning process produces a lot of iron oxide particles called as mill scale. Major components of these particles are wustite (FeO), magnetite ($Fe_3O_4$), and hematite ($Fe_2O_3$). In this study, we tried to produce pure magnetite from the mill scale because of the largest phosphate adsorption capacity of the magnetite. The mill scale was treated with acid (HCl+$H_2O_2$), base (NaOH), and acid-base ($H_2SO_4$+NaOH). Batch adsorption tests showed the acid and/or base treatment could increase the phosphate adsorption capacity of the iron oxides from 0.28 to over 3.11 mgP/g. Magnetite, which could be obtained by acid and base treatment of the mill scale, showed the best adsorption capacity. From the kinetic analysis, both Freundlich and Langmuir isotherm well described the phosphate adsorption behavior of the magnetite. In Langmuir model, maximum phosphate adsorption capacity was found to be 5.1 mgP/g at $20^{\circ}C$.

• Korean Journal of Food Science and Technology
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• v.34 no.2
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• pp.151-156
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• 2002

Five kinds of red wine from three different grape varieties, Gerbong (G), Campbell (C), Moru (M), Gerbong+Moru (70:30, GM) and Gerbong+Campbell (70:30, GC), were prepared. Their chemical and microbiological changes were monitored during alcohol fermentation and aging. The changes of pH, brix and total acidity were $3.2{\sim}3.6,\;17.9{\sim}6.0$ and $2.4{\sim}4.6\;g/L$, respectively. The viable cell numbers of initial, stationary and death phases were $6.0{\times}10^6,\;1.0{\times}10^8$ and $7.0{\times}10^5\;cfu/mL$ during alcohol fermentation. The sugar fermentability, glucose and fructose contents were greatly decreased less than 0.2 g/L, and the final contents of ethanol and sulfur dioxide were $11.4{\sim}12.3%$ and $40{\sim}62\;mg/L$, respectively. The conversion ratios of malic acid to lactic acid were 23% (G), 67% (M), 28% (C), 33% (GM) and 39% (GC). The chemical characteristics of five red wine were significantly different in pH, total acidity, sulfur dioxide and lactic acid contents (p<0.05).

• Korean Journal of Environmental Agriculture
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• v.16 no.4
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• pp.295-303
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• 1997

To investigate the transformation characteristics of nitrogen and carbon from cow manure compost amended in soil under different moisture conditions, dynamics of nitrogen and carbon were determined periodically for 15 weeks of aerobic incubation at room temperature during July${\sim}$November, 1996. Cow manure compost matured with mixing saw dust was amended with the 4 ratios (0, 2, 4, 6%(wt/wt)) in Ap horizon soil, which collected from green house in Yesan, Chungnam. Moisture was controlled with 0.2, 0.3, 0.4, and 0.5 of mass water conte nt (${\theta}$m) to air dried soil, and water loss was compensated at every sampling. During incubation, soil pH was decreased continuously, that was caused by hydrogen generated from nitrification of ammonium nitrogen. And pH became higher with inclining cow manure compost amendment and water treatment, that meaned the increase of mineralization of organic-N to $NH_4\;^+-N$. Total nitrogen was reduced with increasing water content, but total carbon showed the contrast tendency with that of nitrogen. Therefore, C/N ratio slightly decreased in the low water condition (${\theta}$m 0.2) during incubation, but increased continuously in high water condition over ${\theta}$m 0.4. As a result, it was assumed that soil fertility is able to be reduced in the high water content over available water content. Nitrate transformation rate increased lasting in the low water content less than ${\theta}$m 0.3. Itdropped significantly in the first $2{\sim}3$ weeks of incubation over ${\theta}$m 0.4. In particular, nitrate was not detected in ${\theta}$m 0.5 of water content after the first $2{\sim}3$ weeks. In contrast, ammonium transformation was inclined with increasing water treatment. Nitrogen mineralization rate, which calculated with percentage ratio of (the sum of ex.$NH_4\;^+-N$ and $NO_3\;^--N$)/total nitrogen, was continuously increased in the low water content of ${\theta}$m 0.2 and 0.3. But it saw the different patterns in high water content over ${\theta}$m 0.4 that was drastically declined in the initial stage and then gradually inclined . From the above results, nitrogen transformation patterns differentiated decisively in water content between ${\theta}$m 0.3 and 0.4 in soil. Thus, it is very important for the maintain of suitable soil water content to enhance fertility of soil amended with manure compost. However, excess treatment of manure compost might enhance the possibility of contamination of small watershed and ground water around agricultural area.

• KSBB Journal
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• v.17 no.1
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• pp.73-79
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• 2002

The method to optimize the microbial production of ethanol from CO using Clostridium ljungdahlii was developed. The kinetic parameter study on CO conversion with Clostridium ljungdahlii was carried out and maximum CO conversion rate of 37.14 mmol/L-hr-O.D. and $K_{m}$ / of 0.9516 atm were obtained. It was observed that method of two stage fermentation, which consists of cell growth stage and ethanol production stage, was effective to produce ethanol. When pH was shifted from 5.5 to 4.5 and ammonium solution was supplied to culture media as nitrogen source at ethanol production stage, the concentration of ethanol produced was increased 20 times higher than that without shift. Ethanol production from CO in a fermenter with Clostridium ljungdahlii was optimized and the concentration of ethanol produced was 45 g/L and maximun ethanol productivity was 0.75 g ethanol/L-hr.

• Tuberculosis and Respiratory Diseases
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• v.60 no.2
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• pp.187-193
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• 2006

Background : Normal cell proliferation and viability is strongly depends on the availability of metabolic energy and the maintenance of the appropriate adenylate-nucleotide pools. Hypothetically, changes in adenylate kinase (AK) expression could therefore be associated with adaptation to altered growth characteristics or inversely altered growth characteristics of proliferating cells could drive the changes in the metabolic profile. This study investigated whether the expression of either AK1 or a Mycobacterium tuberculosis adenylate kinase mutant which has the same catalytic activity of AK1 could affect the growth rate of slow-growing BCG. Method : Recombinant BCGs, which were cloned the human muscle-type adenylate kinase synthetic gene (AK1) and adenylate kinase mutation gene (AKmtDM) of Mycobacterium tuberculosis into the Mycobacterium/E.coli expression vectors, were constructed. Recombinant BCGs and wild-type BCG were cultured in 7H9 media and the optical density at 600nm was measured at intervals of 2-3 days. Result : There wasn't the growth rate change induced by AK1 or AKmtDM expression in recombinant BCGs. Conclusion : The expression of AK1 or Mycobacterium tuberculosis adenylate kinase mutant in BCG does not affect the growth rate of BCG.

• Journal of Life Science
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• v.15 no.1 s.68
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• pp.118-123
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• 2005

For the expression in Saccharomyces cerevisiae, Bacillus stearothermophilus cyclodextrin glucano­transferase gene (cgtS) in pCGTS (4.8 kb) was subcloned into the surface expression vector, pYD1 (GALl promoter). The constructed plasmid, pYDCGT (7.2 kb) was introduced into S. cerevisiae EBY100 cells, and then yeast transformants were selected on the synthetic defined media lacking tryptophan. The formation of cyclodextrin (CD) was confirmed with active staining of culture broth of transformant grown on starch medium. Enzymatic reaction products with respect to the culture time and the reaction time were examined by TLC analysis. The results indicated that the enzyme activity was exhibited after 12 h cultivation and CD was produced after 10min of enzymatic reaction. When the surface-engineered yeast cells were cultured on galactose medium, maximum activities of CGTase were about 21.3 unit/l and 16.5 unit/l at $25^{\circ}C\;and\;30^{\circ}C$, respectively. The plasmids stability showed about $80\%\;even\;at\;25^{\circ}C\;and\;30^{\circ}C$.

• Proceedings of the Korean Society of Toxicology Conference
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• 2002.05b
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• pp.106.2-132
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• 2002

형질전환 (유전자 결핍; Knockout) Min 마우스를 이용하여 대장암 발생에서 배추, 양배추 주성분인 indole-3-carbinol (I3C)의 대장암 예방효과를 규명하고자 하였다. 실험동물로는 C57BL/6J-Apc$^{min/+}$(Min 마우스)계의 5내지 6주령의 수컷 이형접합체 형질전환 마우스 70마리와 C57BL/6J계의 동일 산자, 동일 주령의 수컷 wildtype 비형질전환 마우스 10kfl를 The Jackson Laboratory 사 (Bar harber, ME, USA)로부터 직접 구입하였다. C57BL/6J-Apc$^{min/+}$계 수컷 이형접합체 형질전환 (Min)마우스 70마리를 각 군 20내지 25마리씩 세군으로 나누었다. Group 1에는 20마리, Group 2에는 25마리, Group 3에는 25마리를 배치하고, I3C 투여 실험군 (Group 1과 2)에는 실험시작시에 AIN-76A 분말사료에 I3C가 각각 100 및 300ppm이 함유되도록 조제하여 공급하였다. 그리고 실험군(Group 3)에는 실험시작부터 종료시까지 AIN-76A 정제고형사료(Teklad사, WI, USA)를 자유로이 급이하였다. 각군간의 체중, 사료 및 음수소비량을 매 2주마다 측정하였고, 10주간 (16주령)의 실험종료시에는 최종체중과 간장, 신장, 비장 등의 장기무게를 측정하여 상대장기 무게비를 산출하였다. 대조군으로서 C57BL/6J계의 동일 산자, 동일 주령의 수컷 wildtype 비형질전환 마우스 10마리는 같은 조건의 사육실에서 AIN-76A 정제고형사료를 33주간 자유로이 급이하였다. 실험동물은 부검전에 하룻밤 동안 절식하고 이산화탄소 흡입 마취하에서 흉대동맥을 절단하여 방혈하고 각 장기(심장, 폐, 위)를 적출하여 생리심염수에 넣어 장기무게를 측정하고 포르말린에 고정하였다. 소장과 대장의 검사를 위하여 위의 식도부위와 직장을 실로 결찰하여 적출하고 생리심염수를 주입하여 팽창시켜, 십이지장, 공장, 및 회장, 그리고 대장으로 나누어 여과지에 펼친 후 포르말린에 고정하였다. 소장과 대장은 육안 및 자동 영상분석길ㄹ 이용한 분석이 끝난 후에 각 부위별로 4-6개의 절편을 작제하여 포르말린에 재고정하고, 통상적인 조직처리과정, 파리핀 포매 및 3-4$\mu$m 두께의 조직절편을 제작하여 H&E 염색을 실시하여 현미경으로 검경하였다. 약 1주일간의 포르말린 고정이 끝난 소장 및 대장을 부위별, 별 종양개수 및 분포를 자동영상분석기(Kontron Co. Ltd., Germany)로 분석하였다. 체의 변화, 장기무게, 사료소비량 및 마리당 종양의 개수에 대한 통계학적 유의성 검증을 위하여 Duncan's t-test로 통계처리 하였고, 종양 발생빈도에 대하여는 Likelihood ration Chi-square test로 유의성을 검증하였다. C57BL/6J-Apc$^{min/+}$계 수컷 이형접합체 형질전환 마우스에 AIN-76A 정제사료만을 투여한 대조군의 대장선종의 발생률은 84%(Group 3; 21/25례)로써 I3C 100ppm 및 300ppm을 투여한 경우에 있어서는 각군 모두 60%(Group 1; 12/20 례, Group 2; 15/25 례)로 감소하는 경향을 나타내었다. 대장선종의 마리당 발생개수에 있어서는 C57BL/6J-Apc$^{min/+}$계 수컷 이형접합체 형질전환 마우스에 AIN-76A 정제사료만을 투여한 대조군은 1.40$\pm$0.24(100%)에 비하여 I3C 저농도 투여 실험군(Group 1; 0.85$\pm$0.23; 61%, P<0.01), 그리고 I3C 고농도 투여 실험군(Group 2 ; 1.32$\pm$0.29 ; 94%)의 순으로 감소하였다. 선종의 크기별 종양의 발생개수의 분포에 있어서 I3C 저농도 투여 실험군에 있어서는 선종의 크기가 3mm이하의 수가 현저하게 감소하였다. C57BL/6J-Apc$^{min/+}$계 수컷 이형접합체 형질전환 마우스에 AIN-76A 정제사료만을 투여한 대조군의 부위별 소장선종의 발생수는 십이지장부위를 제외하고 각 군에서 유의한 변화는 관찰되지 않았다. 십이지장 종양의 발생개수에서만 I3C 저농도 투여 실험군(Group 1 ; 3.11$\pm$0.85)이 대조군 (Group 3: 1.48$\pm$0.35) 및 I3C 고농도 투여 실험군(Group 2: 1.56$\pm$0.47)에 비하여 유의성 있게 증가하였다. (P<0.05). 따라서 I3C은 소장에서는 암예방 효과가 뚜렷하지 않으나, 대장에 대한 암에방 효과가 있을 것으로 생각된다. 소장 및 대장을 제외한 간장, 신장, 비장, 심장, 폐 그리고 위 등의 기타 장기에서의 조직병리학적 변화는 관찰되지 않았다. 소장 및 대장의 종양은 선종(polyps)으로 관찰되었다. 지난 10여년간 형질전환 및 유전자 결핍 실험동물의 종류가 기하 급수적으로 증가하여 이용되고 있다. 가족성 대장 선종성 용종증(FAP)의 대표적인 모델로 이용되고 있는 C57BL/6J-Apc$^{min/+}$계 수컷 이형접합체 형질전환 마우스를 사용하여 배추나 양배추의 주요성분인 Indole-3-carbinol(I3C)의 대장암 예방효과가 있는지를 검색하여 본 결과 AIN-76A정제사료만을 투여한 대조군의 대장선종의 발생률 84%에 비하여 I3C 100 및 300ppm을 투여한 실험군에 있어서 각군 모두 60%로서 감소하는 경향을 나타내었으며, 대장선종의 마리당 발생개수에 있어서는 대조군의 1.40$\pm$1.041를 100%로 환산하였을 경우 I3C 저농도 및 고농도 투여 실험군에서는 각각 약 61%와 94%를 나타내여 감소하였다. 특히 대장선종의 크기별 분포에 있어서 선종의 크기가 3mm이하의 수가 현저하게 감소하였다. 따라서 저농도 I3C의 투여는 실험적 유전성 가족성 대장 선종성 용종증 모델에 있어서 어느정도 암 예방효과가 있는 것으로 생각된다. 그러나 소장 선종의 발생에는 별 영향이 없는 것으로 생각된다. 그러나 본 실험에 사용된 C57BL/6J-Apc$^{min/+}$계 수컷 이형접합체 형질전환 마우스는 실험개시 시점이 7내지 8주령이 경과하여 이미 태생기부터 소장 및 대장의 선종 발생이 진행되어 온 것을 감안하고 특히 비스테로이드계 항염증 소염제(NSAIDS)와 같은 강력한 COX-2억제제가 아님을 고려하면, 상당한 선종의 발생을 억제할 수 있는 가능성이 매우 높다고 생각한다. 또한 이제까지 배추나 양배추 성분의 복합성분들에 대한 실험적 대장암 모델에서의 촉진효과 등에 대한 보고들이 있어 온 점을 고려할 때 위암(Kim 등, 1994) 간암(Kim 등, 1994), 유방암(Grubbs, 등, 1995; Bradlow 등, 1995)에 대한 예방효과가 있을 것으로 생각된다. 앞으로 이러한 종양조직내에서의 COX-2 및 iNOS mRNA와 단백질의 발현정도를 분자병리학적으로 연구중에 있으며, 향후 십자화과식물 성분인 indole-3-carbinol이 마우스뿐 만 아니라 랫드의 화학발암물질에 의한 대장종양에 대한 억제효과 있는지 연구 필요가 있다. Min 마우스와 같은 형질 전환(유전자결핍;knockout) 실험동물을 이용한 새로운 중기 발암성 시험범의 확립을 통한 각종 환경 유해물질의 발암성 유무 및 COX-2 억제작용이 있는 식품인자의 암예방 후보물질을 체계적으로 검색하는데 유용하게 활용될 수 있을 것으로 생각한다.

• Microbiology and Biotechnology Letters
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• v.36 no.1
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• pp.49-54
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• 2008

The gene encoding human pancreatic pro-carboxypeptidase B (CPB) was cloned and fused to Saccharomyces cerevisiae mating factor alpha-1 secretion signal $(MF{\alpha}1)$, in which the transcription of $MF{\alpha}1$-pro-CPB was under the control of GAL10 promoter. The constructed plasmid $pY{\alpha}$-hproCPB(7.72 kb) was transformed into S. cerevisiae 2805. The recombinant human pro-CPB (hproCPB) was successfully expressed in S. cerevisiae after induction of galactose, and could be secreted into the culture medium. By analyses of SDS-PAGE and western blotting, the molecular weight of the purified hproCPB was estimated to be a 45.9kDa. The activity of extracellular hCPB after removal of pro-region by trypsin treatment reached about 10.16 unit/ml at batch culture of S. cerevisiae $2805/pY{\alpha}$-hproCPB for 60 h. Also, the Km value of partially purified recombinant hCPB is about 0.43 mM.