• 미생물학회지
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• 제42권3호
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• pp.195-198
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• 2006

Anthrax lethal toxin은 탄저병의 치사원인이 되는 독소이며, Lethal toxin은 두 종류의 단백질 PA (Protective antigen)과 LF (lethal factor)로 구성되어 진다. PA는 세포표면의 수용체와 결합하여 LF를 세포질 안으로 이동시켜 주는 역할을 한다. LF는 금속 이온$(Zn^{2+})$ 의존적 단백질 가수분해 효소로써 MKKs[MAPK (mitogen-activated protein kinase) kinases] 집단 단백질의 아미노 말단 부분을 절단하여 대상 세포를 죽음으로 유도하는 것으로 알려져 있다. 본 연구에서는 LF에 대한 특성 분석 및 억제제 개발에 과한 연구를 위해 cell-based high-throughtput screens 개발에 선행되어야 하는 기초 자료를 마련하는데 그 목적이 있다. 이를 위하여 LF의 절단 대상이 되는 기질이 MEK1을 yeast내에서 동시 발현시켜 LF의 활성을 검증하였다. 먼저 효모(Saccharomyces cerevisiae)를 숙주로 하여 LF의 기질인 MEK1 발현 vector를 구축하였고, 구축된 발현 system을 기본을 LF 활성을 검증하고자 yeast에 형질전환하여 plasmid의 안전성 및 MEK1 유전자의 발현 및 LF에 의한 MEK1 아미노말단의 절단 부위를 확인하였다. 본 연구는 세포내 검증 system 도입의 기초적 자료를 제공하였으며, yeast내의 MEK1 발현은 탄저병의 저해제 선별 및 활성 측정 검증을 생체에서 고효율적이며, 안정적으로 할 수 있다는 가능성을 나타냈다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권4호
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• pp.275-287
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• 2000

Fermentation strategies for recombinant protein production in Pichia pastoris have been investigated and are reviewed here. Characteristics of the expression system, such as phenotypes and carbon utilization, are summarized. Recently reported results such as growth model establishment, app58lication of a methanol sensor, optimization of substrate feeding strategy, DOstat controller design, mixed feed technology, and perfusion and continuous culture are discussed in detail.

• BMB Reports
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• 제40권1호
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• pp.82-87
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• 2007

Our heterologous expression system of the human ferritin H-chain gene (hfH) allowed us to characterize the cellular effects of ferritin in yeasts. The recombinant Saccharomyces cerevisiae (YGH2) evidenced impaired growth as compared to the control, which was correlated with ferritin expression and with the formation of core minerals. Growth was recovered via the administration of iron supplements. The modification of cellular iron metabolism, which involved the increased expression of high-affinity iron transport genes (FET3 and FTR1), was detected via Northern blot analysis. The findings may provide some evidence of cytosolic iron deficiency, as the genes were expressed transcriptionally under iron-deficient conditions. According to our results examining reactive oxygen species (ROS) generation via the fluorescence method, the ROS levels in YGH2 were decreased compared to the control. It suggests that the expression of active H-ferritins reduced the content of free iron in yeast. Therefore, present results may provide new insights into the regulatory network and pathways inherent to iron depletion conditions.

• 한국산학기술학회논문지
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• 제15권8호
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• pp.5412-5418
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• 2014

참돔 이리도바이러스(RSIV)는 이리도바이러스과에 속하며, 많은 아시아 국가에서 감염성 어류 질병을 유발하여 양식산업에 커다란 경제적 손실을 입히는 바이러스이다. 우리는 최근에 효모표면발현(yeast surface display, YSD)를 사용하여 다양한 해양바이러스를 동정하고 검출할 수 있는 새로운 실험시스템을 개발하였다. 이 연구에서 우리는 참돔 이리도 바이러스(RSIV)의 외피단백질을 효모표면 발현 기법을 이용하여 발현시켰다. 바이러스 외피단백질 유전자는 염기서열 데이터베이스에 기초하여 합성되었고, 효모발현벡터인 pCTCON2으로 서브클로닝되었다. 이 벡터는 효모 strain EBY100으로 형질전환 되었다. Flow cytometry와 Western blot analysis를 통해 RSIV 외피단백질의 발현을 확인하였다. ${\beta}$-mercaptoethanol 처리에 의해 발현된 바이러스 외피단백질을 효모 표면로부터 분리하였다. 이 연구의 결과는 YSD 시스템이 해양바이러스 외피단백질을 획득하기 위한 매우 좋은 발현시스템이라는 것을 보여준다.

• 자연과학논문집
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• 제4권
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• pp.1-10
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• 1991

발현 Vector인 pKK223-3를 이용하여 효모 Thiol-Specific Antioxidant단백질 유전자를 대장균에 도입시켜 이 단백질을 발현시켰다. 이 단백질은 대장균 단백질의 약 1% 정도로 발현되었으며, 물리 및 화학적 특성은 효모의 것과 동일한 특성을 보였다.

• Journal of Microbiology and Biotechnology
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• 제23권7호
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• pp.1031-1040
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• 2013

Candida albicans is a dimorphic fungus that commensally colonizes human mucosal surfaces. The aim of this study was to assess the role of different C. albicans morphologies in inducing pattern recognition receptors (PRRs) and cytokines in macrophages. Macrophages may respond to pathogen-associated molecular patterns via TLR2 and TLR4 by expressing cytokines. The hyphal transition of C. albicans was induced by 20% serum (S), RPMI-1640 (R), or $39^{\circ}C$ culture (H). Macrophages were then challenged with either yeast (Y) or different hyphae cultures of C. albicans, followed by RT-PCR and FACS analysis of PRRs expression. In addition, macrophages were stimulated with either yeast or different hyphae cultures of C. albicans used by RT-PCR and Bio-Plex analysis of cytokines production. Macrophages expressed high levels of TLR4 and dectin-1 after stimulation with Y cells. In contrast, stimulation with H or R cells strongly increased the expression of TLR2 and dectin-2. Stimulation with Y cells significantly enhanced the expression of IL-$1{\beta}$ and weakly increased the expression of IL-6 and IL-12. Stimulation with hyphal cells (S, R, and H) strongly increased IL-10 expression, but weakly reduced IL-$1{\beta}$ expression. The phagocytosis activity and NO production of macrophages were decreased upon treatment with hyphal cells compared with yeast, and depended on the length of hyphae. In summary, the yeast and hyphae forms of C. albicans resulted in an induction of different PRRs, with accompanying differences in immune cell cytokine profiles.

• BMB Reports
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• 제43권2호
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• pp.110-114
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• 2010

The yeast three-hybrid system (Y3H), a powerful method for identifying RNA-binding proteins, still suffers from many false positives, due mostly to RNA-independent interactions. In this study, we attempted to efficiently identify false positives by introducing a tetracycline operator (tetO) motif into the RPR1 promoter of an RNA hybrid expression vector. We successfully developed a tight tetracycline-regulatable RPR1 promoter variant containing a single tetO motif between the transcription start site and the A-box sequence of the RPR1 promoter. Expression from this tetracycline-regulatable RPR1 promoter in the presence of tetracycline-response transcription activator (tTA) was positively controlled by doxycycline (Dox), a derivative of tetracycline. This on-off control runs opposite to the general knowledge that Dox negatively regulates tTA. This positively controlled RPR1 promoter system can therefore efficiently eliminate RNA-independent false positives commonly observed in the Y3H system by directly monitoring RNA hybrid expression.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권1호
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• pp.7-12
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• 2000

The expression of the mouse $\alpha-amylase$ gene in the methylotrophic yeast, P pastoris was investigated. The mouse $\alpha-amylase$ gene was inserted into the multi-cloning site of a Pichi a expression vector, pPIC9, yielding a new expression vector pME624. The plasmid pME624 was digested with SalI or BglII, and was introduced into P. pastoris strain GSl15 by the PEG1000 method. Fifty-three transformants were obtained by the transplacement of pME624 digested with SaiII or BglII into the HIS4locus $(38\;of\;Mut^+\;clone)$ or into the AOX1 locus $(15\;of\;Mut^s\;clone)$. Southern blot was carried out in 11 transformants, which showed that the mouse $\alpha-amylase$ gene was integrated into the Pichia chromosome. When the second screening was performed in shaker culture, transformant G2 showed the highest $\alpha-amylase$ activity, 290 units/ml after 3-day culture, among 53 transformants. When this expression level of the mouse $\alpha-amylase$ gene is compared with that in recombinant Saccharomyces cerevisiae harboring a plasmid encoding the same mouse $\alpha-amylase$ gene, the specific enzyme activity is eight fold higher than that of the recombinant S. cerevisiae.

• Journal of Microbiology and Biotechnology
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• 제24권9호
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• pp.1178-1188
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• 2014

In this study, the yeast Pichia pastoris was genetically modified to assemble minicellulosomes on its cell surface by the heterologous expression of a truncated scaffoldin CipA from Clostridium acetobutylicum. Fluorescence microscopy and western blot analysis confirmed that CipA was targeted to the yeast cell surface and that NtEGD, the Nasutitermes takasagoensis endoglucanase that was fused with dockerin, interacted with CipA on the yeast cell surface, suggesting that the cohesin and dockerin domains and cellulose-binding module of C. acetobutylicum were functional in the yeasts. The enzymatic activities of the cellulases in the minicellulosomes that were displayed on the yeast cell surfaces increased dramatically following interaction with the cohesin-dockerin domains. Additionally, the hydrolysis efficiencies of NtEGD for carboxymethyl cellulose, microcrystal cellulose, and filter paper increased up to 1.4-fold, 2.0-fold, and 3.2-fold, respectively. To the best of our knowledge, this is the first report describing the expression of C. acetobutylicum minicellulosomes in yeast and the incorporation of animal cellulases into cellulosomes. This strategy of heterologous cellulase incorporation lends novel insight into the process of cellulosome assembly. Potentially, the surface display of cellulosomes, such as that reported in this study, may be utilized in the engineering of S. cerevisiae for ethanol production from cellulose and additional future applications.

• Current Research on Agriculture and Life Sciences
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• 제30권1호
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• pp.1-11
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• 2012

We identified cdf based on screening of the Arabidopsis cDNA library for functional suppressors of the AtBI-1 (a gene described to suppress the cell death induced by Bax gene expression in yeast). The cdf was located on Chr. V and was composed of 5 exons and 4 introns. It encodes a protein of 258 amino acid residues with a molecular weight of 28.8 kDa. The protein has 3 transmembrane domains in the C-terminal region. The cdf has one homologue, named cdf2, which was found in Arabidopsis. Like cdf, cdf2 also induced growth defect in yeast. The effect of the cell growth defect factor was somewhat lower than Bax. cdf could arrest the growth of yeast. Its localization to the nucleus was essential for the suppression of yeast cell proliferation. Morphological abnormality of intracellular network, which is a hallmark of AtBI-1, was attenuated by expression of cdf.