• KSBB Journal
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• v.26 no.3
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• pp.189-192
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• 2011

Much interest has been recently focused on the production of large quantities of hydrogen, due to its potential importance in our economy and needs in the petroleum and chemical industries. Formate dehydrogenase H (FDH-H) from Escherichia coli containing selenocysteine that oxidizes formate to carbon dioxide with the release of hydrogen is a component of the anaerobic formate hydrogen-lyase complex of E. coli. To make full use of FDH-H, we need effective expression condition. In this approach, we investigated the effect of pH on FDH-H stability and observed the effect of selenite and formate concentration on the activity of FDH-H. Additionally, coexpression of selenocysteine insertion genes were tried to improve the expression of FDH-H. The highest level of FDH-H expression was achieved by coexpression of selenocysteine insertion genes (pSUABC) as well as by the addition of $10\;{\mu}M$ selenite and 10 mM formate. At this optimized condition, a 2.6 fold elevation of expression of FDH-H was achieved.

• Korean Journal of Veterinary Research
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• v.29 no.3
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• pp.407-416
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• 1989

The prospect of live vaccines consisting of genetically modified vaccinia virus expressing foreign genes is exciting, but important issues concerning safety and efficacy need to resolved. Vaccinia virus (VV) is an efficient expression vector with broad host range infectivity and large DNA capacity. This vector has been particularly useful for identifying target antigens for humoral and cell-mediated immunity. The WHO smallpox eradication program, involving the extensive use of VV vaccines, resulted in the late 1970s in the elimination of one of the world's most feared diseases. This achievement is a triumph for preventive medicine and for international collaboration in public health. In 1980, WHO recommended that the routine use of smallpox vaccine should be stopped. Against this background, the prospect of li ve vaccines consisting of genetically modified VV expressing foreign antigens arising from the work of Moss, and Paoletti and their colleagues in 1982 has been greeted with enthusiasm. These investigators have shown that genes coding for immunogenic proteins can be inserted into VV DNA without impairing the ability of the virus to grow in cell culture. Moreover experimental animals infected with VV recombinants containing genes coding for a variety of immunizing proteins have been shown to be protected against challenge infection with the corresponding infectious agent. In this communication, I describe current progress in the construction of a novel plasmid vector that facilitate the insertion and expression of foreign genes in VV as well as the selection of recombinants.

• Journal of The Korean Society of Grassland and Forage Science
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• v.20 no.2
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• pp.97-102
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• 2000

To increase thennotolerance of forage crops, transgenic rice plants as a model for transformation of monocots were generated. A cDNA encoding the chloroplast-localized small heat shock protein (small HSP) of rice, Oshsp21, was introduced into rice plants via Agrobacterium-mediated gene transfer system. Calli induced from scutella were co-cultivated with a A. tumefaciens strain EHAlOl canying a plasmid, pIGhsp21. A large number of transgenic plants were regenerated on a medium containing hygromycin. Integration of Oshsp2l gene was confirmed by PCR and Southern blot analyses with genomic DNA. Northern blot and immunoblot analyses revealed that the Oshsp21 gene was constitutively expressed and accumulated as mature protein in transgenic plants. Effects of constitutive expression of the OshspZl on thermotolerance were first probed with the chlorophyll fluorescence. Results indicate that inactivation of electron transport reactions in photosystem I1 (PSII), were mitigated by constitutive expression of the Oshsp21. These results suggest that the chloroplast small HSP plays an important role in protecting photosynthetic machinery during heat stress. (Key words : Thermotolerance, Rice, Transgenic, cDNA)

• Korean Journal of Plant Tissue Culture
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• v.27 no.2
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• pp.95-100
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• 2000

The cytosolic glutathione reductase (GR) gene of Brassica campestris L. was introduced into several Japonica cultivars of rice by Agrobacterium tumefaciens and a large number of transgenic plants were produced. Three-week old calli were co-cultivated with A. tumefaciens strain EHA101 carrying the plasmid pIGR1. The efficiency of transformation was differed from rice cultivars. A Japonica cultivar, 'Daeribbyeo' appeared the highest efficiency (42.5%) of transformation among the four cultivars tested. The addition of acetosyringone (50 $\mu$M) during co-cultivation was a key to successful transformation. Transgene fragments were identified by PCR amplification and further confirmed by Southern blot analysis. Mendelian inheritance of the transgenes was confirmed in T$_1$ progeny.

• Journal of Microbiology and Biotechnology
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• v.9 no.5
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• pp.593-603
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• 1999

Poly(3-hydroxybutyrate) (PHB) production by fermentation was examined under both restricted- and ample-oxygen supply conditions in a single fed-batch fermentation. Recombinant Escherichia coli transformed with the PHB production plasmid pSYLl07 was grown to reach high cell density (227 g/l dry cell weight) with a high PHB content (78％ of dry cell weight), using a glucose-based minimal medium. A simple flux model containing 12 fluxes was developed and applied to the fermentation data. A superior closure (95％) of the carbon mass balance was achieved. When the data were put into use, the results demonstrated a surprisingly large excretion of formate and lactate. Even though periods of severe oxygen limitation coincided with rapid acetate and lactate excretion, PHB productivity and carbon utilization efficiency were not significantly impaired. These results are very positive in reducing oxygen demand in an industrial PHA fermentation without sacrificing its PHA productivity, thereby reducing overall production costs.

• BMB Reports
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• v.31 no.2
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• pp.123-129
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• 1998

Foreign proteins, $endo-{\beta}-1,4-glucanase$ of Bacillus subtilis, preS1+S2 region of hepatitis B virus large surface antigen, human ${\beta}_2-adrenergic$ receptor ($h{\beta}_{2}AR$), and bovine growth hormone (bGH) were expressed in Saccharomyces cerevisiae and secreted into the medium. These proteins were expressed using the alcohol dehydrogenase I (ADH1) promoter of Saccharomyces cerevisiae and secreted by signal sequence of the 97 K killer toxin gene of doublestranded linear DNA plasmid (pGKL1) of S. cerevisiae. All these proteins underwent severe modifications; in particular, N-glycosylation in the case of $endo-{\beta}-1,4-glucanase$, $h{\beta}_2AR$, and preS1+S2. Seventy four percent of the expressed $endo-{\beta}-1,4-glucanase$ was secreted into the culture medium. Highly modified proteins were detected in the culture medium and in the cell. Expressed $h{\beta}_2AR$, which has seven transmembrane domains, remained in the cell. The degrees of secretion and modification and the states of proteins in the culture medium and in the cell were quite different. These results indicated that the nature of the protein has a critical role in its secretion and modifications.

• Molecules and Cells
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• v.39 no.9
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• pp.687-691
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• 2016

Transcription activator-like effector nucleases (TALENs) are powerful tools for targeted genome editing in diverse cell types and organisms. However, the highly identical TALE repeat sequences make it challenging to assemble TALEs using conventional cloning approaches, and multiple repeats in one plasmid are easily catalyzed for homologous recombination in bacteria. Although the methods for TALE assembly are constantly improving, these methods are not convenient because of laborious assembly steps or large module libraries, limiting their broad utility. To overcome the barrier of multiple assembly steps, we report a one-step system for the convenient and flexible assembly of a 180 TALE module library. This study is the first demonstration to ligate 9 mono-/dimer modules and one circular TALEN backbone vector in a one step process, generating 9.5 to 18.5 repeat sequences with an overall assembly rate higher than 50%. This system makes TALEN assembly much simpler than the conventional cloning of two DNA fragments because this strategy combines digestion and ligation into one step using circular vectors and different modules to avoid gel extraction. Therefore, this system provides a convenient tool for the application of TALEN-mediated genome editing in scientific studies and clinical trials.

• Applied Chemistry for Engineering
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• v.17 no.5
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• pp.557-560
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• 2006

The epoxide hydrolase (EH) of Aspergillus niger LK was expressed to high levels in Escherichia coli based on codon usage. E. coli, Rosetta (DE3)PLysS, containing a large number of tRNAs for rare-codons was employed as a host strain. The recombinant E. coli expressing A. niger EH showed an enhanced enantioselective hydrolysis activity toward racemic styrene oxide. Enantiopure (S)-styrene oxide with a high enantiopurity of 99% ee was obtained from racemic substrates.

• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.8-15
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• 2021

More and more available fungal genome sequence data reveal a large amount of secondary metabolite (SM) biosynthetic 'dark matter' to be discovered. Heterogeneous expression is one of the most effective approaches to exploit these novel natural products, but it is limited by having to clone entire biosynthetic gene clusters (BGCs) without errors. So far, few effective technologies have been developed to manipulate the specific large DNA fragments in filamentous fungi. Here, we developed a fungal BGC-capturing system based on CRISPR/Cas9 cleavage in vitro. In our system, Cas9 protein was purified and CRISPR guide sequences in combination with in vivo yeast assembly were rationally designed. Using targeted cleavages of plasmid DNAs with linear (8.5 kb) or circular (8.5 kb and 28 kb) states, we were able to cleave the plasmids precisely, demonstrating the high efficiency of this system. Furthermore, we successfully captured the entire Nrc gene cluster from the genomic DNA of Neosartorya fischeri. Our results provide an easy and efficient approach to manipulate fungal genomic DNA based on the in vitro application of Cas9 endonuclease. Our methodology will lay a foundation for capturing entire groups of BGCs in filamentous fungi and accelerate fungal SMs mining.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.4
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• pp.317-324
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• 2003

목 적: 본 실험의 목적은 자궁내막세포를 분리 및 배양법 확립과 함께 불멸화 시키는 것이다. 방 법: 자궁내막에서 상피세포(epithelial cells)와 기질세포(stromal cells)의 분리는 Satyawaroop 등(1979)의 방법에 기초를 두었다. 자궁내막에서 상피세포와 기질세포의 순수 분리도를 확인하고, 불멸화된 기질세포에서 SV40 large T antigen을 확인하기 위하여 면역형광 염색(immunocytochemistry)과 Western blot 기법을 이용하였다. 정상 기질세포의 경우 subconfluence (60%) 상태에서 transfection을 진행하였다. 순수 분리된 plasmid DNA와 Qiagen 사의 superfect를 이용하여 transfection을 실시하였다. 결 과: 본 연구에서 우리는 두 가지 형태의 자궁내막 세포의 분리 및 배양에 성공하였다. 상피세포는 다면체의 형태를 띠며, 선(grandular)조직의 조각으로부터 나선형으로 자란다.기질 세포는 길쭉한 형태를 띠며, 상피세포에 비해 오래 살고, 빠르게 증식하여 나란한 형태로 배열된 세포 다발(cell bundle)을 형성한다. 이렇게 분리된 세포들은 95%의 균질성을 보였으며, 면역형광염색과 western blot을 통해 확인 하였다. 한편 SV40(Simian Virus 40) large T 항원을 암호화 하고 있는 염기 서열을 포함한 플라스미드 벡터로 안정적인 트랜스펙션을 시킴으로써 불멸화 된 자궁내막의 기질 세포주를 확립하였다. 불멸화 된 세포는 그 세포가 유래한 정상의 세포와 동일한 표현형을 가지고 있었다. 결 론: 본 연구에서, 우리는 자궁내막에서 상피세포(epithelial cells)과 기질세포(stromal cells)를 분리하여 배양법을 확립하였다. 동시에 SV40 large T antigen을 이용하여 불멸화된 세포주를 확립하였다. 이렇게 확립된 세포주는 자궁의 생리작용 연구 및 자궁내막증(Endometriosis)과 자궁암(Endometrial cancer) 등과 같은 여러 자궁관련 질병 연구에 많은 도움이 될 것으로 사료된다.