• BMB Reports
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• 제30권1호
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• pp.80-84
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• 1997

The synthesis and secretion of human angiogenin in E. coli by the natural leader sequence has been studied. We constructed a recombinant plasmid containing human angiogenin cDNA which encompassed all the coding region including leader sequence required for secretion. The recombinant plasmid was introduced into a suitable E. coli host. The angiogenin was detected in the culture medium and periplasm upon the induction of gene expression. The molecular weight of the secreted angiogenin was identical to that of authentic angiogenin purfied from human plasma when estimated by SDS-PAGE and immunoblotting. showing that the natural leader sequence was recognized and processed by the secretion machinery of E. coli. The angiogenin concentration in the culture medium reached a maximum within 2 h when expressed at $37^{\circ}C$ with 0.02~2 mM IPTG. In contrast, the expression level increased gradually over time up to 11 h at $23^{\circ}C$ with 0.002~2 mM IPTG and at $37^{\circ}C$ with 0.002 mM IPTG.

• 산업기술연구
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• 제28권B호
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• pp.59-63
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• 2008

Rapid production of therapeutic proteins such as angiostatin and endostatin angiogenic inhibititors has been highly demanded for cancer treatment. In this regard, recombinant human angiostatin and endostatin were successfully expressed as soluble forms by maltose binding protein (MBP)-mediated fusion expression in Escherichia coli. PCR amplified, angiostatin and endostatin genes from human placenta cDNA library were inserted into an expression vector pMAL-c2e to construct prokaryotic expression vectors, pMAL-c2e/AS and pMAL-c2e/ES, respectively. Recombinant angiostatin and endostatin were efficiently expressed in E. coli origami (DE3) after IPTG induction and protein expression were confirmed by SDS-PAGE analyses. The expressed recombinant proteins were purified near homogenity using an amylose affinty column chromatography. In contrast that previous E. coli expressions were all insoluble, our results first time demonstrated that MBP fused human angiostatin and endostatin were soluble in E. coli.

• Food Science and Biotechnology
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• 제15권5호
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• pp.817-819
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• 2006

8-Quinolinol and other quinolinol derivatives were evaluated with regard to their growth-inhibitory effects against intestinal bacteria, using the paper disk-agar diffusion method. The observed growth responses varied according to the chemicals and dosages used, as well as the bacterial species tested. 8-Quinolinol showed a significant inhibitory effect against Clostridium difficile, C. perfringens, and Escherichia coli, at 5, 2, 1, and 0.5 mg/disk, and also exhibited a very strong inhibitory effect at 0.25 mg/disk. At low concentrations, 8-quinolinol had strong inhibitory effects against C. perfringens at 0.1 and 0.05 mg/disk; 8-quinolinol also manifested a moderate inhibitory effect against C. perfringens at 0.025 mg/disk. Furthermore, 8-quinolinol revealed moderate and weak growth inhibition against C. difficile and E. coli at concentrations of 0.1 and 0.05 mg/disk, respectively, but 2-quinolinol, 4-quinolinol, and 6-quinolinol evidenced no growth inhibition against B. bifidum, B. longum, C. difficile, C. perfringens, E. coli, or L. casei. The inhibitory effects of 8-quinolinol against C. difficile, C. perfringens, and E. coli lead to its consideration as a possible therapeutic modality for the treatment of diseases associated with harmful intestinal bacteria.

• 한국식품위생안전성학회지
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• 제13권1호
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• pp.1-5
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• 1998

본 연구는 E. coli O157:H7에 대한 LP system(lactoperoxidae/thiocyanate/hydrogen peroxide)의 항균효과를 측정하기 위해 수행되었다. 초기 접종수준($10^{2},\;10^{4},\;10^{7}cfu/ml$), LP의 농도 (10ppm, 20ppm, 30ppm), 배지종류 (TSB, UHT milk, raw milk), 배양온도 ($5^{\circ}C,\;10^{\circ}C,\;15^{\circ}C$) 등에 따라 E. coli O157:H7에 대한 항균효과를 측정, 비교한 결과, 초기 접종수준을 $10^2/ml$으로 하였을 때와 LP의 농도를 10ppm 및 $5^{\circ}C$ 배양에서 항균력이 가장 높게 나타났다.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.216-219
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• 2004

The effect of culture temperature on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) in recombinant E. coli was investigated. E. coli cell was cotransformed with two plasmids (pTCGT1 and pGroll) in which the cgt and groEL/ES genes are under the control of T7 promoter and pzt-1 promoter, respectively. When tetracycline (10 ng/ml) and IPTG (l mM) were added as inducers at the early-exponential phase (2 h) and mid-exponential phase (3h), respectively, the solubilization of the inclusion body CGTase was greatly dependent on the temperature of the culture. At low culture temperature of $25^\circ{C}$, 2- or 3-fold higher activity and specific activity were obtained over $37^\circ{C}$. SDS-PAGE analysis revealed that about 62% of CGTase in the total CGTase protein was found in the soluble fraction by applying overexpression of GroEL/ES chaperone and by cultivation of E. coli at $25^\circ{C}$, whereas 33% of CGTase was detected in the soluble fraction at $37^\circ{C}$. Therefore, the expression of GroEL/ES and cultivation at $25^\circ{C}$ greatly enhanced the soluble production of CGTase in E. coli.

• KSBB Journal
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• 제29권4호
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• pp.244-249
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• 2014

Biosynthesis pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHA) from fatty acid ${\beta}$-oxidation pathway was constructed in recombinant Escherichia coli by introducing the Pseudomonas sp. 61-3 PHA synthase gene (phaC2) and the maoC genes from Pseudomonas putida, Sinorhizobium meliloti, and Ralstonia eutropha. The metabolic link between fatty acid ${\beta}$-oxidation pathway and PHA biosynthesis pathway was constructed by MaoC, which is homologous to P. aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1). When the E. coli W3110 strains expressing the phaC2 gene and one of the maoC genes from P. putida, Sinorhizobium meliloti, and Ralstonia eutropha were cultured in LB medium containing 2 g/L of sodium decanoate as a carbon source, MCL-PHA that mainly consists of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD), was produced. The monomer composition of PHA and PHA contents varied depending on MaoC employed for the production of PHA. The highest PHA content of 18.7 wt% was achieved in recombinant E. coli W3110 expressing the phaC2 gene and the P. putida maoC gene. These results suggest that MCL-PHA biosynthesis pathway can be constructed in recombinant E. coli strains from the b-oxidation pathway by employing MaoC able to supply (R)-3-hydroxyacyl-CoA, the substrate of PHA synthase.

• BMB Reports
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• 제40권1호
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• pp.107-112
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• 2007

To study the functioning of HSP70 in Escherichia coli, we selected NtHSP70-2 (AY372070) from among three genomic clones isolated in Nicotiana tabacum. Recombinant NtHSP70-2, containing a hexahistidine tag at the amino-terminus, was constructed, expressed in E. coli, and purified by $Ni^{2+}$ affinity chromatography and Q Sepharose Fast Flow anion exchange chromatography. The expressed fusion protein, $H_6NtHSP70$-2 (hexahistidine-tagged Nicotiana tabacum heat shock protein 70-2), maintained the stability of E. coli proteins up to 90$^{\circ}C$. Measuring the light scattering of luciferase (luc) revealed that NtHSP70-2 prevents the aggregation of luc without ATP during high-temperature stress. In a functional bioassay (1 h at 50$^{\circ}C$) for recombinant $H_6NtHSP70$-2, E. coli cells overexpressing $H_6NtHSP70$-2 survived about seven times longer than those lacking $H_6NtHSP70$-2. After 2 h at 50$^{\circ}C$, only the E. coli overexpressing $H_6NtHSP70$-2 survived under such conditions. Our NtHSP70-2 bioassays, as well as in vitro studies, strongly suggest that HSP70 confers thermo-tolerance to E. coli.

• 한국식품위생안전성학회지
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• 제32권1호
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• pp.64-69
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• 2017

본 연구에서는 양상추에 접종된 E. coli O157:H7, S. Typhimurium과 L. monocytogene에 대하여 저온 플라즈마와 UV-C 단독처리 및 병행처리 효과를 측정하였다. E. coli O157:H7, S. Typhimurium, L. monocytogenes는 양상추에 초기 농도가 5.82, 5.09, 5.65 log CFU/g이 되도록 각각 접종하였다. 저온 플라즈마와 UV-C를 처리된 양상추는 $4^{\circ}C$에서 9일간 보관하며 미생물학적 분석과 관능평가를 실시하였다. 저온 플라즈마 처리는 E. coli O157:H7, S. Typhimurium, L. monocytogenes의 개체 수를 각각 0.26, 0.65, 0.93 log CFU/g 수준으로 감소시켰다. 또한, UV-C 처리 시 각각 0.87, 0.88, 1.14 log CFU/g 수준으로 감소되었다. 또한 UV-C 처리 후 저온 플라즈마를 처리한 병행처리에서는 각각 1.44, 2.70, 1.62 log CFU/g 수준으로 감소하였다. 저온 플라즈마와 UV-C 단독처리 보다는 병행처리가 좀 더 효과적으로 균을 저감하는 것으로 판단되었다. 관능평가 결과는 외관, 질감, 전체적인 수용도 면에서 대조구와 비교하였을 때, 보관 6일까지 유의적인 차이가 없었다. 따라서 저온 플라즈마와 UV-C 병행처리는 양상추에 존재하는 균을 저감하기 위한 효과적인 기술로 활용될 수 있을 것으로 생각되었다.

• 한국식품과학회지
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• 제34권6호
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• pp.1134-1139
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• 2002

Campylobacter, Arcobacter, Helicobacter는 분류학적으로 동일한 rRNA superfamily Ⅵ로 식중독 이외에도 위궤양, 위암, 유산 및 신경 장애를 유발한다. Campylobacter, Arcobacter, Helicobacter를 오염된 식품 등에서 선택적으로 검출하기 위해 PCR, multiplex-PCR, RFLP(restriction fragment length polymorphism)의 기법을 이용하였다. Campylobacter, Arcobacter, Helicobacter의 16S rRNA를 target으로 하는 CHA primer를 사용하여 동일한 PCR product의 검출할 수 있었다. C. jejuni와 C.coli를 A. butzleri와 H. pylori로부터 선택적으로 검출하기 위해 fla A gene을 target으로 하는 pg3, p50을 사용하였으며, A. butzleri는 23S rRNA를 target으로 하는 Arco2, Butz를 이용했다. 또한 H. pyloyi는 isocitrate dehydrogenase gene을 target으로 하는 icd1, icd2를 사용하였고, C. jejuni는 ceuE gene을 target으로 하는 JEJ1, JEJ2를 이용하여 효과적으로 분리검출이 이루어졌다. 또한 제한효소 Dde I 을 사용하여 PCR-RFLP를 통해 C. jejuni, C. coli를 A. butzleri, H. pylori로부터 분리할 수가 있었다. 따라서 이러한 primer를 이용하여 C. jejuni, C. coli, A. butzleri, H. pylori가 함께 오염되었을 때 각각 균주의 선택적인 검출이 가능할 것이다.

• BMB Reports
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• 제38권1호
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• pp.97-103
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• 2005

Under the condition of expression of $\lambda$ P protein at lethal level, the oriC DNA-binding activity is significantly affected in wild-type E. coli but not in the rpl mutant. In purified system, the $\lambda$ P protein inhibits the binding of both oriC DNA and ATP to the wild-type DnaA protein but not to the rpl DnaA protein. We conclude that the $\lambda$ P protein inhibits the binding of oriC DNA and ATP to the wild-type DnaA protein, which causes the inhibition of host DNA synthesis initiation that ultimately leads to bacterial death. A possible beneficial effect of this interaction of $\lambda$ P protein with E. coli DNA initiator protein DnaA for phage DNA replication has been proposed.