콩단백질은 글리시닌과 β-콘글리시닌을 주요 성분으로 한다. 영양성 및 가공 특성을 개선하기 위하여 유전자공학적인 방법을 시도하였다. 즉 β-콘글리시닌의 β-서브유니트롤 유전자 클로닝하고 대장균에서 발현시켰다. 발현벡타는 pET 21d이며 플라스미드를 구축하여 E.coli BL21(DE3)에 형질전화시켰다. 발현된 단백질은 균체 전체 단백질의 20%이며 가용화 상태로 축적되었다. 축적 발현단백질은 천연의 β-콘글리시닌과 동일항 트러머로 확인되었다. 정제는 호아산암모늄 20∼40%분별침전, Q-Sepharose 이온교환크로마토그라피, Butyltoyoperal 소수성 컬럼크로마토그라피로 하였다. 이것은 콩단백질의 특성을 규명하는데 필요한 대장균 대량 발현계를 확입하고 발현단백질의 정제방법을 확립한 결과이다.

Bacillus thuringiensis subsp. kurstaki HD-1으로부터 생산된 살충성 내독소 단백질을 coding하는 CryIIA 유전자를 클로닝하고 염기서열을 조사하였다. HD-1 균주의 12개 plasmid 중 225kb plasmid를 분리하여 CryIIA 유전자를 포함하는 5kb HindIII 절편을 hybridization하여 찾아냈다. 이 절편을 plasmid pUC19에 ligation하여 E. coli에 형질 전환하였다. 이 독소 유전자를 포함하는 4kb BamHI-HindIII 절편은 vector pT7-5에 ligation하여 pSKIIA라하였다. pSKIIA는 3개의 open reading frames(orf1, orf2, orf3)로 구성되어 있으며 염기서열은 3,952base로 되어 있었다. 이러한 3개의 orf 각각의 발현 여부를 확인하기 위하여 생물검정을 하였다. 그러나 orf1 또는 orf2에 의한 형질 전환체는 독성이 없는 것으로 나타났다. orf3를 포함하는 형질 전환체는 3종의 나비목 곤충(배추점나방, 담배거세미나방, 담배나방) 및 1종의 파리목 곤충(집모기) 유충에 대하여 독성을 나타내었다.

한국산 멧누에(Bomdyx mandarina)chorion 유전자의 형질 발현기구를 규명하기 위한 연구의 일환으로 본 실험을 실시했다. 멧누에의 난각구조를 주사형 전자현마경에 의해 관찰한 결과 매우 특이적인 구조가 인정되었다. 즉 원추상의 불규칙 돌기에 의한 돌기구조층과 이 돌기 구조층을 덮고 있는 한 층의 얇은 덮개 구조층이 그것이다. 2차원 전기영동법에 의해 chorion 단백질을 분석한 결과, 난각을 구성하는 주요 단백질 성분은 등전점 4~6, 분자량 6~30 kd로 밝혀졌다. 특히 특이난각구조와 관련된 특이단백질 성분을 검출하였으며 이들의 대부분은 고cysteine 단백질인 것으로 추정했다. 이상의 연구결과에 의해 멧누에 난각의 특이 구조 형성에 따른 유전자발현기구 규명을 위한 기초자료가 얻어졌다.

Water temperature is one of the most important factors of fish survival, affecting the habitat, migration route, development, and reproduction. This experiment studied the induction level of heat shock protein (HSP70) mRNA and protein in a walleye pollock (Gadus chalcogrammus) primary hepatocyte culture based on different temperatures. Hepatocytes were attached at 7.5 °C for 24 hours. Hsp70 induction levels were then measured for 48 hours at 5, 8, 11, 14, and 17 °C. The induction level was lowest at 5 °C and generally increased with temperature until 14 °C. The induction level was reduced at 17 °C, indicating that 14 °C is the highest tolerable temperature for hepatocytes. These data indicate that primary hepatocyte cell culture is under no stress at 5 and 8 °C. Temperatures greater than 11 °C induce stress, showing similar induction patterns in both mRNA and protein in hepatocytes. The results suggest that 14 °C is the maximum internal defense temperature of walleye pollock survival.

Background: Prenatal exposure to infectious and/or inflammatory insults can increase the risk of developing neuropsychiatric disorder such as bipolar disorder, autism, and schizophrenia later in life. We investigated whether Valeriana fauriei (VF) treatment alleviates prepulse inhibition (PPI) deficits and social interaction impairment induced by maternal immune activation (MIA).Methods and Results: Pregnant mice were exposed to polyriboinosinic-polyribocytidilic acid (5㎎/㎏, viral infection mimic) on gestational day 9. The adolescent offspring received daily oral treatment with VF (100㎎/㎏) and injections of clozapine (5㎎/㎏) for 30 days starting on the postnatal day 35. The effects of VF extract treatment on behavioral activity impairment and protein expression were investigated using the PPI analysis, forced swim test (FST), open field test (OFT), social interaction test (SIT), and immunohistochemistry. The MIA-induced offspring showed deficits in the PPI, FST, OFT, and SIT compared to their non MIAinduced counterparts. Treatment with the VF extract significantly recovered the sensorimotor gating deficits and partially recovered the aggressive behavior observed in the SIT. The VF extract also reversed the downregulation of protein expression induced by MIA in the medial prefrontal cortex.Conclusions: Our results provide initial evidence of the fact that the VF extract could reverse MIA-induced behavioral impairment and prevent neurodevelopmental disorders such as schizophrenia.

The roots of Platycodon grandiflorum species either dried or fresh, are used as an ingredient in salads and traditional cuisine in Korea. To interpret the root proteins, a systematical and targeting analysis were carried out from diploid and tetraploid roots. Two dimensional gels stained with CBB, a total of 39 differential expressed proteins were identified from the diploid root under in vivo condition using image analysis by Progenesis Same Spot software. Out of total differential expressed spots, 39 differential expressed protein spots (≥ 1.5-fold) were analyzed using LTQ-FTICR mass spectrometry. Except two proteins, the rest of the identified proteins were confirmed as down-regulated such as Isocitrate dehydrogenase, Proteasome subunit alpha type-2-B. However, the most of the identified proteins from the explants were mainly associated with the oxidoreductase activity, nucleic acid binding, transferase activity and catalytic activity. The exclusive protein profile may provide insight clues for better understanding the characteristics of proteins and metabolic activity in various explants of the economically important medicinal plant Platycodon grandiflorum.

Soybean is very useful crop to supply vegetable protein for human. However, cultivation arear of this economically important crop is gradually diminished in upland field. Hence, cultivation area of soybean is increased in paddy field. During the growth duration of soybean, excessive moisture injury is serious problem for sustainable production and supply. We investigated protein expression according to different period of seed soaking and germination after seed soaking. For comparison on expression of protein according to different condition, we performed two-dimensional electrophoresis. After electrophoresis analysis, we selected differentially expressed protein spots according to different condition such as soaking period and germination after soaking to identify protein function by using MALDI-TOF. Results revealed that pattern of expression of protein according to soaking period and germination after soaking were generally not different in major spots. However, degree of expression of protein in some protein spots was increased in accordance with decrease of soaking period. Especially, in Hwangkeum-Kong, Danyeop-Kon, and Pecking, the degree of expression of protein was remarkably increased for 4 days after soaking. But, according to germination after soaking, degree of expression of protein in germinated seeds of all cultivars was higher than un-germinated seeds. In results of MALDI-TOF analysis, specific proteins were identified by different soaking period such as Allergen Gly m Bd 28K, P24 oleosin isoform B. Also, in accordance with germination, degree of protein expression of the related protein, Gibberellin was increased in un-germinated seeds of Iksan-Kong. In ungerminated seeds of Sinpaldal-kong, proteins were identified as down-regulated by soaking such as ATP binding and Inhibitor II', proteinase.

In the present study, different expression of protein from Taekwang was revealed by 2-DE, and expressions of protein on each week after flowering was investigated. After analysis of expression of protein, MALDI-TOF was executed to identify expected protein function. Results revealed that there were three patterns of expression of protein during the maturing. The first pattern was that proteins were gradually expressed as up-regulation from 1 week to 6 week. The second pattern was that proteins were expressed gradually from 1 week to 5 week and then it started down-regulation in 6 week. The last pattern was that proteins were gradually as up-regulation from 1 week to 3 week and then down-regulation until 6 week. This phenomenon suggests that young stage has more protein related to correspondence mechanism against disease and growth and then maturing stage has more expression of protein related to storage protein. In MALDI-TOF analysis, p24 oleosin isoform A protein was identified that relates oleosin which is synthetic product in oil body. This protein spot increased gradually until 5 week and then decreased after 5 week. It explained that the protein is active until maturing stage to protect oil in seed and then its activity has gradually degraded. This result may be expected that a protein, related to growth of a seed has increased until maturing and then a seed fills up with a storage protein

Soybean is very useful crop to supply vegetable protein for human. Supply of soybean is increased because it has useful ingredient. Recently, cultivation of soybean in paddy field is increasing due to the increase of rice stockpile in Korea. Hence, in this study, expression of protein was identified regarding different environment for cultivation to investigate the effect of different environment on protein expression. Two-dimensional electrophoresis was performed to investigate the expression of protein using image analysis program to measure degree of protein expression in numerical value. Hannam-kong, Beakcheon-Kong, Hwangkeum-Kong, and Danwon-Kong were used as plant material. 2-DE combined with image analysis revealed that each degree of protein expression of Hannam-Kong and Hwangkeum-Kong in upland field was higher than degree of protein expression in paddy field. However, in case of Beackcheon-Kong, the phenomenon was opposite. In Danwon-kong, the degree of protein expression was not different between up-land field and paddy field. To this end, major protein spots were not different between paddy field and upland field among all cultivars. It could be suggested that protein expression is not severely different by various environment, but different environment affects degree of protein expression.

This study was conducted to improve the postharvest storage techniques of managing and storing seeds, totest qualities and viabilities of the seeds and to examine the germination rate and the protein expression of Achyranthesjaponica Nakai. The seeds collected from different areas of Je-Cheon and Gwang-Ju were stored with different temperaturesand durations. Two plant growth regulators and two seed priming were treated to investigate their effect on the germinationrates and the days required for germination. The weight of one hundred seed collected in Gwang-Ju was heavier than thosein Je-Cheon. Seed length collected in Gwang-Ju was also longer about 5.12㎜ than those in Je-Cheon about 4.90㎜ andseed width was longer in Gwang-Ju than those in Je-Cheon. The rates of seed germination in two different collection areaswere higher about 2.9 to 13.0% in Gwang-Ju compared to those in Je-Cheon. Comparing its rates with the storing tempera-tures and durations, they were not clearly different in between 4℃ and 25℃ and they also were gradually decreased withgetting longer storing durations. The germination rates treated by plant growth regulators were higher with GA3 than thosewith Kinetin. The highest seed germination rate was appeared at 50 ppm of GA3. Comparing its rates with different seedpriming, they were relatively higher with KNO3 than those with PEG6000. In protein expression patterns between before thegerminating and after the germinating of seeds, more and clear bands were appeared in the seed after the germination com-pared to those before the germination of seeds, especially 10~20kDa. These results showing more and clear bands weremore clearly appeared in Gwang-Ju compared to Je-Cheon. Comparing the protein expression with plant growth regulatorsand seed primings, GA3 was better expression than those with Kinetin and KNO3 was better than those with PEG6000. Moreand clear bands were closely related to the germination rates of seeds and more detailed studies would be required.

This study was conducted to investigate the quality of seeds, the germination rates and the days required for germination, to examine the patterns of protein expressions during the germination and to improve the techniques of managing and storing seeds and viability of the seeds of Lithospermum erythrorhizon Sieb. et Zucc. After collecting and harvesting seeds, they were classified to white and brown colors of seed coat through testing their seed size, weight, and quality. The germination rates, the days required for germination, and the protein expressions were examined with different colors of seed coats, storing temperatures and durations by treating the different plant growth regulators and primings. One hundred seed weight of white color was heavier about 1.17 g than those of brown one about 0.81 g. The germination rates in white color of seed coat was higher, 3.05 ~ 5.75%, than those in brown one. Its rates were decreased with getting longer in storage durations. There was no big differences on germination rates between storage temperatures. The plant growth regulator of GA3 and Kinetin was affected to improve the seed germination. GA3 increased the seed germination clearly at 25 ppm level, while kinetin increased it gradually from 25 to 100 ppm levels. In germination by seed primings, PEG6000 made higher germination rate with increasing their levels, whereas KNO3 increased the germination until 100 mM level and then decreased it with 200 mM unlike PEG6000. The protein expressed during the seed germination were appeared more and clearer bands in the seed after germination, especially 20 ~ 30 kDa, compared to those in the seed before germination. These results showing more and clearer bands were positively related to the germination rates which were different by seed colors, storage temperatures and durations, and plant growth regulators and primings.

Protein arginine methyltransferase (PRMT) family 단백질은 히스톤 H3의 arginine 잔기를 메틸레이션 시켜줌으로써 chromatin remodeling을 유발하여 다양한 유전자의 전사를 활성화시켜 주는 것으로 알려져 있다. Coactivator-associated arginine methyltransferase 1 (CARM1/PRMT4)이 그 중의 하나로, 이는 발생과정에서 폐세포로의 분화에 관여하는 것으로 알려져 있으며, 세포의 종류에 따라서 지방세포와 근육세포로의 분화에 영향을 줄 뿐만 아니라, 생쥐 배아줄기세포의 전분화능 유지에도 관여한다는 연구 보고가 있다. 그러나 CARM1이 인간 중간엽 줄기 세포 (hMSCs)에 어떤 영향을 미치는지에 대해서는 현재까지 알려진 바가 없다. 따라서 본 연구에서는 인간 중간엽 줄기세포에 CARM1 단백질을 직접 도입함으로써 변화되는 유전자의 발현 양상을 관찰하였다. 먼저, CARM1 단백질에 세포침투단백질 (cell-penetrating peptide) 서열을 접합시킴으로써, CARM1 단백질이 효율적으로 세포 내로 도입되도록 하였으며, 그 결과 CARM1 단백질이 6시간 내에 충분히 세포막을 통과하여 세포질 및 핵 내로 이동하며, 24시간 내에는 대부분의 단백질이 핵 내에 존재하는 것을 관찰하였다. 또한 도입된 CARM1 단백질이 히스톤 H3의 arginine 잔기를 메틸화시키는 것을 확인하였다. 이렇게 순수분리 정제된 재조합 CARM1 단백질의 처리 후, chromatin remodeling이 일어난 인간 중간엽 줄기세포의 유전자 발현 변화 양상을 microarray를 통해 확인한 결과, 지방세포, 골세포, 근육세포, 신경세포, 그리고 췌장세포 분화에 관여하는 전체 유전자의 약 35～45%의 유전자에서 발현양상의 변화가 나타났다. 따라서 본 연구는 재조합 CARM1 단백질의 세포 내 직접 도입이 CARM1 유전자의 도입으로 우려되는 문제들을 보완하면서도 CARM1 단백질 본연의 기능을 효과적으로 수행한다는 것을 보여주며, 생체 친화적 단백질에 의한 인간 중간엽 줄기세포의 유전자 발현 양상의 변화를 통해 그의 분화와 관련한 임상적 이용의 가능성을 보다 높일 수 있다는 점을 시사한다.

Estrogen related receptor β(Esrrb)는 오르판 수용체 중 하나로 전분화능 관련유전자인 Oct4와 Nanog의 발현을 조절함으로써 줄기세포의 미분화를 유지시키고, 지속적인 자기 복제를 가능케 하는 유전자로 알려져 있다. 또한 Feng 등 (2009)은 체세포에 Oct4, Sox2와 함께 Esrrb 유전자를 함께 도입하면, 유전자가 변형된 체세포가 배아 줄기세포와 유사한 유도만능줄기세포로 리프로그래밍(reprograming)되어 진다는 결과를 보고한 바 있다. 본 연구에서는 인간 ESRRB 단백질을 양수유래줄기세포 내로 직접도입하는 방법을 개발하고, 이를 통해 전분화능 관련유전자의 기능 조절을 확인하고자 하였다. 클로닝 된 인간 short-form ESRRB를 세포투과 펩타이드(cell-penetrating peptide, CPP)의 일종인 R7(아르기닌 7개)에 접합(Fusion)하였고, 합성단백질 (R7-ESRRB-His6)의 형태로 배양중인 인간 양수 유래 줄기세포에 처리하여 세포내로 도입하였다. R7-ESRRB-His6 단백질은 5시간 내에 세포막을 통과하였고, 24시간 내에 핵 내로 이동하였다. 또한 핵 내로 이동한 ESRRB 단백질은 OCT4와 NANOG 유전자의 발현을 증가시켰을 뿐만 아니라, 또 다른 전분화능 관련유전자인 SOX2의 발현도 함께 증가시킨다는 것을 확인하였다. 이상의 결과는 세포투과 펩타이드와 유전자의 접합을 통해 생산된 R7-ESRRB-His6 합성단백질이 양수유래줄기세포내로 원활하게 도입되는 것을 확인하였고, 유전자의 변형 없이 전분화능 관련유전자의 기능을 조절할 수 있는 방법임을 확인하였다.

Liriope platyphylla has been though as an useful medical plant to improve the cough, sputum, neurodegenerative disorders, obesity, and diabetes in Korea and China from old times. In order to investigate the effects of Liriope platyphylla on expression and secretion of nerve growth factor (NGF), the mRNA expression and protein secretion were detected in the neuronal cell (B35) and neuroglial cell (C6) cultured with three differences concentration (5%, 10%, 15%) of Liriope platyphylla. In MTT assay and FACS anslysis, the some death of some B35 and C6 cells were observed in 15% extract-treated group, while other groups did not induce the death. Also, the mRNA expression of NGF were significantly increased in 5% and 10% extracts treated-group. Furthermore, the NGF protein concentration in supernatant collected from cultured cells showed the very similar pattern with mRNA expression. In order to verify the activity of secreted NGF, the culture supernatant collected from B35 and C6 cells cultured with Liriope platyphylla extracts for 24 hrs were treated into undifferentiated PC12 cells, and the differentiation level of PC12 cell were also observed with microscopes. The differentiation level of PC12 cell were significantly increased depend on the dose of extract. Therefore, these results suggested that the water extracts of Liriope platyphylla may contribute the regulation of NGF expression and secretion in the neuronal cell and be considered as an excellent candidate for a neurodegenerative disease-therapeutic drug.