The baculovirus expression vector system (BEVS) is an effective and widely used method for the production of recombinant proteins in insect cells or larvae. However, the expression efficiency of foreign proteins using the polyhedrin promoter could not obtain the protein yields observed for native polyhedrin. To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of various polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Among the fusion-expressed protein in nucleus and cytoplasm, the most hyper-expression was observed in the fusion of amino acids 19 to 110 and 32 to 59 of polyhedrin. Additionally, the several proteins expressed by the partial polyhedrin-fused expression system was markedly increased. However, we identified that hyper-expression of target protein varied depending on the partial polyhedrin. Therefore, we constructed the virus inducible partial polyhedrin fusion transient expression system. This system amenable for screening of suitable partial polyhedrin to produce the target protein. The present study suggests a new option for higher expression of useful foreign recombinant protein using the partial polyhedrin fusion expression in baculovirus.

A novel oxidant fumigation (NOF) is a commercial bleaching and disinfection agent. Recent study indicates its insecticidal activity. However, its exact mode of action to kill insects is not known. This study sets up a hypothesis that reactive oxygen species released from NOF is a main factor to kill insects. Plodia interpunctella is a lepidopteran insect pest infesting various stored grains. Both larvae and adults were susceptive to NOF. To test the hypothesis, we needed to identify antioxidant genes in P. interpunctella. Superoxide dismutase (SOD) and thioredoxin-peroxidase (Trx) were identified from P. interpunctella EST library using ortholog sequences of Bombyx mori. Both SOD and Trx were expressed in larvae of P. interpunctella expecially against oxidative stress induced by bacterial challenge. The bacterial challenge also induced some heat shock protein (HSP) genes. Similarly, different doses of NOF significantly induced both SOD and Trx genes. There results suggest that NOF at sublethal doses releases reactive oxygen species, which may be detoxified by the antioxidant activities of SOD and Trx of P. interpunctella.

1. Pepsin과 pancreatin 소화물들을 비교한 결과, 순수한 팥 단백질 중에서 소화효소 저항성을 가지는 단백질이 존재하는 것으로 확인되었다. 2. 팥의 주요 단백질을 제거하고 pepsin과 pancreatin으로 소화시켰을 때 더 많은 분해가 일어나는 것으로 보아 팥의 주요단백질들 중에 소화효소 저항성을 가지는 것이 많을 것이라 추측된다. 3. 팥의 주요 단백질들은 장 점막세포와는 크게 작용하지 않는 것을 확인할 수 있었다. 4. 팥 단백질의 데이터베이스 구축과 팥 단백질이 다른 영양소들과의 상호작용을 하는 지에 대한 연구가 진행되어야 할 것이다.

Early onset torsion dystonia is caused by mutations in DYT1 gene in humans. Two deletion mutations and one missense mutation were found from patients with this devastating disorder. The molecular and cellular etiology underlying this disorder is not still understood yet. Because vertebrates have more than 4 homologs in their genomes, it is very hard to elucidate the exact in vivo functions of Torsin1A. Instead, Drosophila has only one homolog named Torsin. To investigate the in vivo functions of Torsin, we generated and characterized transgenic flies expressing coding regions of Torsin mRNA or double stranded inhibitory DNA constructs (RNAi). The specific antibodies for Drosophila Torsin (DTor) also were generated. The transgenic expression of DTor cDNA or RNAi in all tissue induced significant changes in DTor proteins levels. Even though expression of DTor cDNA in neuronal system increased the amount of DTor proteins, expression of DTor RNAi did not significantly altered the amount of DTor. Consistent with this result, the numbers of flies with motor-activity were not discernible among neuronal expression lines. However, flies expressing DTor cDNA or RNAi on muscles showed significantly altered locomotor ability, suggesting that DTor plays important roles in regulating motor-activity at the post-synaptic terminals of motor neurons. In addition, DTor over-expressing flies showed increased resistance to H2O2. In the future study, we will found how those phenotypes were accomplished by performing various experiments. (NRF-2012R1A1A4A01011674: HRF-S-201.-6)

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of various polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the previously reported minimal region for self-assembly and the KRKK nuclear localization signal (NLS). The marked increase of EGFP by these fusion expressions was confirmed through protein and fluorescence intensity analyses. Among the fusion-expressed protein in nucleus and cytoplasm, the most hyper-expression was observed in the fusion of amino acids 19 to 110 and 32 to 59 of polyhedrin. Also these fragments, some degradation of only the fused polyhedrin was observed in the fusion of amino acids 19 to 85 and 32 to 85. The production of E2 protein, which is a major antigen of classical swine fever virus, was dramatically increased by fusion expression with polyhedrin amino acids 19 to 110, and its preliminary immunogenicity was verified using experimental guinea pigs. The production of luciferase was approximately two folds increased by fusion expression with polyhedrin amino acids 32 to 59, and its activity was measured using Luminometer. This study suggests a new option for higher expression of useful foreign recombinant protein using the partial polyhedrin fusion expression in baculovirus.

To profile the proteome in porcine plasma, blood samples were collected from adult male barrows and those plasma were retrieved. For the depletion or pre-fractionation of high-abundance proteins, plasma samples were treated with commercial kits. Then, protein profiling was initiated using one and two-dimensional electrophoresis. Proteins were spotted and then identified by MALDI-TOF-TOF and LC-MS-MS. In the results, more than forty six proteins were identified and the reference map was constructed. The pre-treatment for the removal of high-abundance proteins caused the changes in 2-DE images and some of the proteins were newly uncovered after the most of high abundant proteins were removed. However, it is expected for further steps necessary to identify more low-abundance proteins that may contain potential bio-markers.

Proteinaceous insecticidal proteins, Cry proteins, from Bacillus thuringiensis (Bt) are insecticidal proteins that are highly active against several species of Lepidoptera. Thus, cry genes encoding these Cry proteins have been widely applied for construction of transgenic crops resistant to pest insects. In this study, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Spodoptera exigua and Ostrinia furnacalis were identified. We used site-directed mutagenesis to improve the insecticidal activity of Mod-Cry1Ac, resulted 31 mutant cry genes. These mutant cry genes encodes potent insecticidal proteins in the form of crystalline protoxins of 95 kDa. SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. When the insecticidal activities of these mutant Cry proteins against to larvae of P. xylostella, S. exigua and O. furnacalis were assayed, they showed higher or similar insecticidal activity compared to those of Cry1Ac and Cry1C. Especially, Mutant-N16 is considered to have the potential for the efficacious biological insecticide since it showed the highest insecticidal activity.

Early onset torsion dystonia is caused by mutations in DYT1 gene in humans. The molecular and cellular etiology underlying this disorder is not still understood yet. Because vertebrates have more than 4 homologs in their genomes, it is very hard to elucidate the exact in vivo functions of Torsin1A. Instead, Drosophila has only one homolog named Torsin. To investigate the in vivo functions of Torsin, we generated and characterized transgenic flies expressing coding regions of Drosophial Torsin (DTor) cDNA or double stranded inhibitory DNA constructs (RNAi). The transgenic expression of DTor cDNA or RNAi in all tissue induced significant changes in DTor proteins levels as well as ability of motor controls. In addition, DTor over-expressing flies showed increased resistance to H2O2 or paraquat. In the future study, we will found how those phenotypes were accomplished by performing various experiments.

Pulsed electronic field(PEF) 처리에 의한 우유 단백질과 물리화학적 특성의 변화를 확인하기 위하여 원유, 탈지유, HTST, LTLT, UHT 우유를 PEF 처리하였다. 시료 중의 단백질을 SDS-PAGE로 확인하였을 때, PEF 처리에 의한 우유 단백질의 변성은 관찰할 수 없었다. Differential scanning calorimetry(DSC)로 우유 단백질의 열변성 정점 온도(Td)를 분석한 결과, 탈지유를 65oC에서 PEF 처리하였을 때 Td가 87.66oC에서 97.18oC로 증가하여 PEF 처리가 우유 단백질의 변성에 영향을 미치는 것을 확인하였다. PEF 처리에 의한 alkaline phosphatase, protease, lactoperoxidase의 잔존효소활성을 측정한 결과, 원유와 탈지유에서 alkalinephosphatase는 PEF 처리에 의해 효소활성이 감소하였다. 또한 protease와 lactoperoxidase의 활성은 PEF 처리에 의해 영향을 받지 않았다. 65oC에서 PEF 처리한 원유는 처리하지 않은 원유보다 높은 갈색도를 나타내었으나, 기타 우유는 PEF에 의한 유의적인 차이가 없었다. 우유를 PEF 처리하였을 경우 산도의 변화는 관찰되지 않았고 pH의 경우에도 PEF 처리 여부에 따라 유의적인 차이는 있었으나 크게 변화하지는 않았다.

Nuts are one of the most common sources of allergies in individuals of all ages. In order for a particular protein to render an allergic reaction, it must resist proteolytic digestion by intestinal enzymes. In this study, three well-known allergenic nuts, almonds, cashew nuts, and peanuts, were used as samples, and enzyme digestion with Bacillus protease and porcine pepsin was tested. A proteomic approach using two-dimensional gel electrophoresis and an MS/MS analysis was applied to visualize and identify the proteins that were resistant to enzyme digestion. Among the 150 protein spots tested, 42 proteins were assigned functions. Due to the lack of genomic databases, 41% of the identified proteins were grouped as hypothetical. However, 12% of them were well-known allergens, including AraH. The remainder were grouped as storage, enzymes, and binding proteins.

Vitamin C (ascorbic acid) is an essential nutrient of most living tissues. We established a strain of Gulo-/- mice with known deficiency, in which vitamin C intake can be controlled by diet, like humans, and investigated the differen- tially expressed proteins following treatments with Helicobacter pylori and diethylnitrosamine (DENA) in the liver of Gulo-/- mice using a proteomic approach. Expression of p53, 14-3-3ε and 14-3-3δ in Gulo-/- mice liver tissue was analyzed by immunohistochemistry. 2-DE maps constructed from Gulo-/- mice liver and differentially expressed proteins in liver tissue were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/MS). In Gulo- /- mice after H. Pylori infection, followed by treatment with DENA, no differences in p53, 14-3-3ε and 14-3-3δ were observed by immunohistochemistry. Proteome analyses us- ing MALDI-TOF/MS resulted in successful identification of 12 proteins (nine proteins were up-regulated and three were down-regulated). Specifically, peroxiredoxin-6 and Alpha-1-antitrypsin 1-4 were up-regulated in liver after H. Pylori infection followed by treatment with DENA. These results indicated that oral supplementation with vitamin C led to rescue of Gulo-/- mice from vitamin deficiency, and protected the liver from H.pylori infection and/or DENA ef- fect, and vitamin C also protected the liver against oxidative stress.

제초제 성분인 phosphinothricin을 분해하는 phosphinothricin acetyltransferase (PAT) 단백질을 과발현시킨 제초제저항성 감 자가 개발되었다. 본 연구에서는 안전성평가 기준에 따라, PAT 단백질과 제초제저항성 감자에 대한 급성경구독성 및 아만성 독성 평가를 실시하였다. PAT 단백질을 재조합 대장균에서 분리정제 한 후, ICR계 쥐에 2,000 mg/kg weight 용량의 PAT 단백질을 먹이고 14일 동안 치사율과 체중증가를 관찰한 결과, 죽은 동물이 없었고, 체중증가, 사료 섭취 및 특별한 이상증상을 발견하지 못했다. 아만성 독성의 경우 제초제저항성 감자를 표준사료에 0, 1, 5% 섞어 90일 동안 경구 투여하였다. 이 경우에서도 사망한 개체는 없었으며, 조사한 여러 항목에 대해 특별한 이상증후를 관찰하지 못했다. 또한, 제초제저항성 감자를 먹인 동물군과 대조구로 일반감자를 먹인 동물 사이에서 유의 할 만한 차이는 발견되지 않았다. 이런 결과를 토대로 볼 때, 제초제저항성 감자와 제초제 저항성 단백질인 PAT는 동물에게 독소로 작용하지 않음을 확인하였다.

종실 돌연변이를 유기하여 미질개선을 위한 육종으로의 적극적인 활용을 목적으로 얻어진 신동진벼 돌연변이 계통의 작물학적 특성과 종실 저장단백질을 변이모본과 비교 분석을 검토하여 얻은 결과를 요약하면 다음과 같다. 1. 종실 변이계통의 작물학적 특성은 변이모본보다 생태특성의 출수기에서는 대부분 조생의 경향을 보였고, 간장 및 수장은 짧은 경향을 나타냈고, 또한 종실특성의 현미 길이, 현미폭 및 천립중에서도 변이모본보다 짧거나 낮은 정도를 나타냈다. 2. SDS-PAGE 분석결과 opaque 군의 SM-22와 giant embryo 군의 SM-34는 글루테린 폴리펩타이드에서 높은 농도를 보였고, floury 군의 SM-23, shrunken 군의 SM-26, sugary 군의 SM-31은 전단백질 농도패턴은 낮게 보이면서 55kDa 이상의 고분자 band에서 다양성을 나타냈다.

Internalization and expression of extracellular molecules into cells and tissues is known very important process to biological processes and therapy of various diseases. In this study, we analyzed expression pattern of extracellular molecule after transduction into various human cells. To investigate cellular expression of internalized molecule, we used adenovirus containing green fluorescence protein. After infection of adenovirus into various human cells, the efficiency of intracellular gene expression was assessed with determining GFP expressing cells by fluorescence microscopy or FACS. After one day of adenovirus infection into HepG2 and A549, we observed that GFP expression was low at 10moi but expression levels were increased at 100moi in both cells. But, adenovirus infection into HCT116 showed low expression of GFP at concentrations from 1moi to 100moi. After 2 day infection with adenovirus, GFP expression level at 10moi and 100moi was highly increased in HepG2 and A549 compared with 1 day infection. Especially, GFP expression was significantly increased in HCT116 after 2 days infection. However, GFP expressing SKOV3 cells by adenovirus infection were not found in all the experimental conditions tested. For quantitative analysis of GFP expression of cells by adenovirus infection, we carried out FACS analysis. As a result, GFP was expressed at very low levels at 1moi in all cells used in this experiment. GFP expression slightly increased after increasing moi to 10 in HepG2, HCT116, and A549 cells. By 100moi infection of adenovirus, GFP expression was elevated to 10 fold higher than 10moi in HepG2 and A549 and about 4 fold elevation was observed in HCT116. A549 showed 20 fold higher expression of GFP than SKOV3. We also found that GFP expression by adenovirus infection was the highest in HepG2 cells. Protein expression was enhanced by increasing concentrations or time of adenovirus infection. In these results, GFP expression efficiency of adenoviral gene transduction reveals the highest in HepG2 and lowest in SKOV3 among the cells tested. Taken together, we could confirm that intracellular protein expression efficiency by transduction of extracellular gene was different in various human cells. Our study suggests that the cell types and cellular properties should be carefully examined to enhance expression efficiency of extracelluar molecules in biological research and disease therapy