• 미생물학회지
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• 제48권2호
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• pp.66-72
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• 2012

Nup188 단백질은 진화적으로 보존된 가장 큰 핵공단백질 중의 하나로 핵공복합체의 inner ring을 구성하는 인자이다. Nup188의 이종상동체인 분열효모 S. pombe의 Nup184 단백질은 영양분이 풍부한 완전배지(YES 배지)에서 정상적인 생장과 mRNA의 핵에서 세포질로의 이동에 필요하다. 본 연구에서는 ${\Delta}nup184$ 결실돌연변이를 YES 배지에서 배양할 때 보이는 생장지체와 mRNA export 결함을 상보하기 위해서 Nup184의 카르복시 부위(아미노산 잔기482에서 1628까지)가 필요함을 알아내었다. 또한 이 부위는 GFP-Nup184 융합단백질이 핵막에 위치하기 위해서도 필요하였다. 이 과정에서 S. pombe GeneDB (Sanger 연구소, 영국)에 등록되어 있는 Nup184의 열린읽기틀 (1564개의 아미노산 잔기로 된 단백질로 예측)이 우리가 얻은 염기서열 데이터에 비해 66개의 아미노산 잔기가 짧다는 것을 발견하였다. 이 카르복시-말단 부위는 Nup184의 기능에 반드시 필요하였다. 이외에도 Nup184 단백질이 세포 안에서 SUMO 변형되어 있음을 보였다.

• 미생물학회지
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• 제41권4호
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• pp.300-305
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• 2005

Corynebacterium glutamicum에서 promoter-probe vector인 pSK1Cat을 이용해 분리된 프로모터를 함유하는 단편들 중 가장 높은 활성을 나타낸 $P_{19}$ 단편에 대한 심도 있는 분석을 수행하였다. Subcloning을 실시하여 프로모터 활성을 지닌 DNA 영역을 180 bp로 압축할 수 있었고 $(P_{180})$, 이를 C. glutamicum의 균주개량 측면에서 그 활용성을 분석하였다. C. glutamicum에서 메치오닌 생합성에 관여하는metX유전자의 메치오닌에 의한 repression을 해제시키기 위하여 metX유전자의 promoter를 $P_{180}$ promoter로 교체하였고 $(P_{180}-metX)$, $P_{180}-metX$를 C. glutamicum에 도입하여 발현되는 homoserine acetyltransferase 활성을 다양한 성장조건에서 측정하였다. MB 영양배지에서 배양하는 경 우 $P_{180}-metX$를 함유는 균주는 wild type보다 약 24배 높은 homoserine acetyltransferase 활성을 나타내었다. Tac 프로모터에 연계하는 경우 $(P_{tac}-metX)$, 약 13배의 활성 증가만이 관찰되었다. 최소배지에서 배양한 후 분석한 결과, $P_{180}-metX$에서의 발현양상은 배지에 첨가된 methionine에 의해 영향받지 앓음을 확인하였는데, 이는 $P_{180}$ 단편이 생합성 유전자의 derepression에 의한 아미노산 생산균의 개량에 효율적으로 이용될 수 있음을 의미한다. $P_{180}-metA$를 라이신 생산균에 도입하는 경우 최대 약 0.8g/l의 메치오닌이 생산됨을 확인하였다.

• The Plant Pathology Journal
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• 제35권4호
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• pp.351-361
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• 2019

In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant $G05{\Delta}phz{\Delta}prn::lacZ$ and E. coli S17-1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a vfr gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on P. fluorescens, however, it was reported that the deletion of the vfr caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the vfr-knockout mutant $G05{\Delta}vfr$ and $G05{\Delta}phz{\Delta}prn::lacZ{\Delta}vfr$. By quantifying ${\beta}-galactosidase$ activities, we found that deletion of the vfr decreased the prn operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant $G05{\Delta}vfr$, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in P. chlororaphis G05.

• 한국가축번식학회지
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• 제24권4호
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• pp.429-437
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• 2000

본 연구는 retrovirus vector system 에 의해서 EPO와 EGFP 유전자가 전이된 소 태아세포를 이용하여 핵치환된 소 난자에서의 이들 유전자의 성공적인 도입을 조사하였다. Non-starved 소 태아세포는 탈핵된 소 난자의 위란강내로 주입되었다. 소 태아 세포와 난자는 세포간 전기자극에 의해 융합시켰으며, 이후 calcium ionophore와 6-dimethylaminopurine를 이용하여 난활성을 유도하였다. 핵치환에 의해 재구성된 난자는 8일 동안 CRlaa 배양액에서 소 난관상피세포와 함께 공배양하였다. 핵치환에 의해 재구성된 187개와 210(EPO, EGFP)개의 소 난자 중에서, 149개와 158(EPO : 80.0%, EGFP : 75.2%)개의 난자가 분할되었고, 이들 분할된 난자 중 36개와 35(EPO : 24.2%, EGFP : 22.2%)개의 난자가 배반포까지 발달하였다. 이들 배반포에서, EPO 유전자는 PCR에 의해 36개의 모든 난자에서 삽입을 확인하였고, EGFP 유전자의 발현은 형광현미경 하에서 35개의 모든 난자에서 확인하였다. 이 결과는 외래유전자가 삽입된 소 태아 세포를 이용하여 핵치환된 난자는 배반포까지 성공적으로 발달할 수 있다는 것을 나타낸다. 더우기, 이러한 방법은 효율적인 형칠전환 소를 생산하는데 이용될 수 있으리라 사료된다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권5호
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• pp.303-310
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• 2002

Cell therapy applied to wound healing or tissue regeneration presents a revolutionary realm to which principles of gene engineering and delivery may be applied. One promising application is the transplantation of cells into the wounded tissue to help the tissue repair. However, when cells are transplanted from in vitro to in vivo, immune rejection occurs due to the immune response triggered by the activation of T-cell, and the transplanted cells are destroyed by the attack of activated T-cell and lose their function. Immune suppressant such as FK506 is commonly used to suppress immune rejection during transplantation. However, such kind of immune suppressants not only suppresses immune rejection in the periphery of transplanted cells but also suppresses whole immune response system against pathogenic infection. In order to solve this problem, we developed a method to protect the desired cells from immune rejection without impairing whole immune system during cell transplantation. Previously, we reported the success of constructing glomerular epithelial cells for removal of immune complex, in which complement receptor of type 1 (CR1) was over-expressed on the membrane of renal glomerular epithelial cells and could bind immune complex of DNA/anti-DNA-antibody to remove immune complex through phagocy-tosis [1]. Attempting to apply the CR1-expressing cells to cell therapy and evade immune rejection during cell transplantation, we constructed three plasmids containing genes encoding a soluble fusion protein of cytolytic T lymphocyte associated antigen-4 (CTLA4Ig) and an enhanced green fluorescent protein (EGFP). The plasmids were transfected to the above-mentioned glomerular epithelial cells to express both genes simultaneously. Using the clone cells for cell transplantation showed that mice with autoimmune disease prolonged their life significantly as compared with the control mice, and two injections of the cells at the beginning of two weeks resulted in remarkable survivability, whereas it requires half a year and 50 administrations of proteins purified from the same amount of cells to achieve the same effect.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.427-433
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• 2008

The cephabacins produced by Lysobacter lactamgenus are ${\beta}$-lactam antibiotics composed of a cephem nucleus, an acetate residue, and an oligopeptide side chain. In order to understand the precise implication of the polyketide synthase (PKS) module in the biosynthesis of cephabacin, the genes for its core domains, ${\beta}$-ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), were amplified and cloned into the pET-32b(+) expression vector. The sfp gene encoding a protein that can modify apo-ACP to its active holo-form was also amplified. The recombinant KS, AT, apo-ACP, and Sfp overproduced in the form of $His_6$-tagged fusion proteins in E. coli BL21(DE3) were purified by nickel-affinity chromatography. Formation of stable peptidyl-S-KS was observed by in vitro acylation of the KS domain with the substrate [L-Ala-L-Ala-L-Ala-L-$^3H$-Arg] tetrapeptide-S-N-acetylcysteamine, which is the evidence for the selective recognition of tetrapeptide produced by nonribosomal peptide synthetase (NRPS) in the NRPS/PKS hybrid. In order to confirm whether malonyl CoA is the extender unit for acetylation of the peptidyl moiety, the AT domain, ACP domain, and Sfp protein were treated with $^{14}C$-malonyl-CoA. The results clearly show that the AT domain is able to recognize the extender unit and decarboxylatively acetylated for the elongation of the tetrapeptide. However, the transfer of the activated acetyl group to the ACP domain was not observed, probably attributed to the improper capability of Sfp to activate apo-ACP to the holo-ACP form.

• Journal of Microbiology and Biotechnology
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• 제26권1호
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• pp.190-196
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• 2016

In this study, the Brucella abortus ohr gene coding for an organic hydroperoxide resistance protein (Ohr) was cloned into a maltose fusion protein expression system (pMAL), inserted into Escherichia coli, and purified, and its immunogenicity was evaluated by western blot analysis using Brucella-positive mouse sera. The purified recombinant Ohr (rOhr) was treated with adjuvant and injected intraperitoneally into BALB/c mice. A protective immune response analysis revealed that rOhr induced a significant increase in both the IgG1 and IgG2a titers, and IgG2a reached a higher level than IgG1 after the second and third immunizations. Additionally, immunization with rOhr induced high production of IFN-γ as well as proinflammatory cytokines such as TNF, MCP-1, IL-12p70, and IL-6, but a lesser amount of IL-10, suggesting that rOhr predominantly elicited a cell-mediated immune response. In addition, immunization with rOhr caused a significantly higher degree of protection against a virulent B. abortus infection compared with a positive control group consisting of mice immunized with maltose-binding protein. These findings showed that B. abortus rOhr was able to induce both humoral and cell-mediated immunity in mice, which suggested that this recombinant protein could be a potential vaccine candidate for animal brucellosis.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.79.2-80
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• 2003

To better understand plant defense responses against pathogen attack, we identified the transcription factor-encoding genes in the hot pepper Capsicum annuum that show altered expression patterns during the hypersensitive response raised by challenge with bacterial pathogens. One of these genes, Ca1244, was characterized further. This gene encodes a plant-specific Type IIIA - zinc finger protein that contains two Cys$_2$His$_2$zinc fingers. Ca1244 expression is rapidly and specifically induced when pepper plants are challenged with bacterial pathogens to which they are resistant. In contrast, challenge with a pathogen to which the plants are susceptible only generates weak Ca1244 expression. Ca1244 expression is also strongly induced in pepper leaves by the exogenous application of ethephon, an ethylene releasing compound. Whereas, salicylic acid and methyl jasmonate had moderate effects. Pepper protoplasts expressing a Ca1244-smGFP fusion protein showed Ca1244 localizes in the nucleus. Transgenic tobacco plants overexpressing Ca1244 driven by the CaMV 355 promoter show increased resistance to challenge with a tobacco-specific bacterial pathogen. These plants also showed constitutive upregulation of the expression of multiple defense-related genes. These observations provide the first evidence that an Type IIIA - zinc finger protein, Ca1244, plays a crucial role in the activation of the pathogen defense response in plants.

• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.797-804
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• 2000

The human immunodeficiency virus type 1 (HIV-1) Tat is one of the viral gene products essential for HIV replication. The exogenous Tat protein is transduced through the plasma membrane and then accumulated in a cell. The basic domain of the Tat protein, which is rich in arginine and lysine residues and called the protein transduction domain (PTD), has been identified to be responsible for this transduction activity. To better understand the nature of the transduction mediated by this highly basic domain of HIV-1 Tat, the Green Fluorescent Protein (GFP) was expressed and purified as a fusion protein with a peptide derived from the HIV-1 Tat basic domain in Escherichia coli. The transduction of Tat-GFP into mammalian cells was then determined by a Western blot analysis and fluorescence microscopy. The cells treated with Tat-GFP exhibited dose- and time-dependent increases in their intracellular level of the protein. the effective transduction of denatured Tat-GFP into both the nucleus and the cytoplasm of mammalian cells was also demonstrated, thereby indicating that the unfolding of the transduced protein is required for efficient transduction. Accordingly, the availability of recombinant Tat-GFP can facilitate the simple and specific identification of the protein transduction mediated by the HIV-1 Tat basic domain in living cells either by fluorescence microscopy or by a fluorescence-activated cell sorter analysis.

• 대한바이러스학회지
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• 제29권3호
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• pp.145-153
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• 1999

We have now constructed a novel recombinant baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) producing polyhedra with Bacillus thuringiensis (Bt) CryIAc crystal protein. The recombinant polyhedra produced by the recombinant baculovirus, Btrus, in insect cells was characterized. The recombinant baculovirus has two independent transcription units in opposite orientations with two promoters, p10 or polyhedrin gene promoter each initiating transcription of either native polyhedrin or fusion protein with polyhedrin and Bt Cry1Ac crystal protein. Surprisingly, this recombinant baculovirus stably produced recombinant polyhedra which were nearly similar to those of wild-type AcNPV. The immunogold staining experiment showed that the recombinant polyhedra were assembled with polyhedrin and Bt Cry1Ac crystal protein, and contained virus particles. Insecticidal toxicity of recombinant polyhedra of Btrus to the fall webworm, Hyphantria cunea, was strikingly improved in comparison with the wild-type AcNPV.