Antioxidant and Anticancer Activities of Platycodon Grandiflorum A. De Candolle Root

이지영 Graduate School of Life Sciences & Biotechnology K 2004 국내박사

한국산 도라지는 오랫동안 전통 한약재로 사용되거나 식용으로 널리 사용되어 왔다. 본 논문에서는 한국산 도라지(4 년근)의 생리활성 검색을 위해 도라지를 석유에텔로 추출하여 얻은 분획을 가지고 항산화 및 항암 활성을 측정한 후, 각 활성물질을 분리, 정제 및 동정하여 분석하기까지의 과정을 기술하였다. 먼저 도라지의 석유에텔 추출물을 실리카겔 컬럼크로마토그래피를 통해 석유에텔과 에틸에텔을 9:1-5:5 (v/v) 비율로 분리하여 총 5 개의 분획 (fraction I-V)을 얻었다. 분획의 항산화 활성 측정은 크게 과산화지질 형성억제능력과 자유 라디칼 소거능력 측정으로 나누었는데, 먼저 과산화지질 형성억제효과를 측정하기위해 Ferric thiocyanate test 와 Thiobarbituric acid test를 행한 결과, 다른 분획보다 FII(8:2)분획에서 높은 과산화지질 형성억제효과를 보였고, 기존의 항산화제와 비교하였을 때 BHA 보다는 못하지만 α-tocopheol 보다 활성정도가 높게 나타났다. 또한 자유라디칼 소거효과(free radical scavenging)를 측정한 결과에서도 FII(8:2) 분획이 다른 분획에 비해 높은 활성을 보였으며 농도에 따라 효과가 증가하는 경향을 나타내었다. 많은 식물에서 밝혀진 상당수의 항산화 물질이 페놀성 물질로 밝혀졌기 때문에 각 분획에서 페놀성 물질 함량을 조사하였으며, 그 결과, FII(8:2) 분획에서 4.80 ±0.26mg/g 으로 다른 분획보다 높게 나타났다. 이를 보다 더 명확하게 분리하기 위해 preparative TLC (thin layer chromatography)를 통해 FII 를 FII-1∼5 분획들로 분리하여 각각의 자유 라디칼 소거능을 측정한 결과, 높은 활성을 보이는 분획을 얻을 수 있었다. 최초 분획들의 항암활성 측정에서는 인체결장암세포인 HT-29, 직장암세포인 HRT-18 과 간암세포인 HepG2 를 대상으로 MTT assay 방법을 통해 각각 분획들의 세포증식억제 효과를 실험한 결과 다른 분획에 비해 FIII(7:3) 분획이 가장 효과적인 활성을 나타내었다. 이것을 역시 Preparative TLC 를 통해 FIII-1∼6 의 분획들로 분리하여 활성을 조사하였고, 특히 FIII-2 에서 가장 높은 활성과 농도 의존적인 세포사멸효과를 관찰하였다. 이 실험을 통해 도라지의 석유에텔 추출물에서 기존의 항산화제와 비교해 높은 활성을 가진 항산화 물질과, 암세포에 따라 약간의 차이가 있으나 항암효과를 나타내는 물질의 존재를 확인하고, 이것은 각각 계속되는 실험을 통해 활성물질의 분리, 정제 후, 동정 및 분석이 이루어졌다. 항산화 물질 연구에서는 앞에서 가장 높은 항산화 효과를 보였던 FII(8:2) 분획을 실리카겔 컬럼 크로마토그래피를 통해 재정제하였고, 그 중 가장 높은 활성을 보이는 FII-3 분획을 HPLC 를 사용하여 높은 항산화 효과를 보이고 서로 비슷한 구조를 가지는 것으로 추측되는 2 개의 물질을 분리, 정제하였다. 이들을 UV spectra, HRFAB-MS, FT-IR 그리고 ^(13)C, ^(1)H-NMR 을 통해 구조를 분석한 결과, 두개의 물질은 페놀성 물질과 지방산이 에스테르 결합으로 연결된 wax esters 임이 밝혀졌다. 두개의 물질 모두 페놀성 부분으로 coniferyl alcohol 을 가지고 있으며 연결된 지방산으로서 compound 1 (분자량 418)이 palmitic acid, compound 2 (분자량 444)가 oleic acid 의 esters 형태로 밝혀졌다. 분리, 정제된 compound 1, 2를 가지고 DPPH 자유 라디칼 소거능을 측정한 실험에서, compound 2 가 1 보다 좀 더 높은 활성을 나타내었고 이는 합성 항산화제인 BHA 보다도 높은 것이었다. 또한 superoxide 와 nitric oxide 자유라디칼 소거능 실험에서도 compound 1, 2 모두 높은 항산화 활성을 보여, BHA 와 BHT 보다 효과적임을 확인하였다. 이러한 페놀성 wax ester 는 보통 식물의 cuticle 이나 suberin 층에서 발견되었으나, coniferyl alcohol 을 페놀부분으로 가지는 wax ester는 지금까지 거의 보고된 바가 없고, 또한 그것의 식물체내 역할 및 생리활성에 대해서도 거의 알려진 바가 없다. 따라서 이 실험을 통해 처음으로 도라지에서 wax ester 물질이 분리되었고 이것의 항산화 활성이 확인되었다. 항암효과를 가지고 있는 물질에 대한 연구에서는, 처음에 분리한 분획 중 항암활성이 좋았던 FIII(7:3)분획을 가지고 preparative-TLC 및 HPLC 를 통해 1개의 활성물질을 분리 및 정제하였다. 이 물질은 추가적인 구조분석이 이루어지지 않았으나, 분리과정에 있어서 UV 흡수 스펙트럼을 통해 전형적인 polyacetylene 구조를 가지고 있음을 확인하였다. 이 물질을 가지고 결장암세포인 HCT-116 세포를 대상으로 실험한 결과, 뚜렷한 세포성장억제의 효과를 보였으며, 20, 40 그리고 60 μg/mL 의 농도와 24, 48, 72 시간 배양한 결과, 시간과 농도 의존적인 활성을 가지고 있음을 확인하였다. 특히 60 μg/mL 의 농도로 처리한 군에서 24시간 이내에 이미 50% 이상의 세포사멸율을 나타내었고, 72 시간 배양 후에는 약 90 %에 가까운 효과를 나타내었다. 이러한 세포성장억제 또는 사멸에 관련되는 세포의 사멸 메커니즘(apoptosis)을 살펴 보기 위해, 인체 결장암세포인 HCT-116을 분리된 활성물질과 함께 배양하였을 때 apoptosis 시 발생하는 세포 형태 및 DNA 의 특징적인 변화여부를 관찰하였다. 세포 모양 및 성장 관찰실험에서 HCT-116 에 활성물질을 처리 후 시간에 따라 세포가 수축되고 세포수도 감소되는 것을 관찰하였고, acridine orange / ethidium bromide 염색 후 세포핵의 변화관찰에서도 세포핵의 분획화와 apoptotic body 로 형성되는 것을 관찰하였는데, 이들은 모두 세포의 apoptosis 중 일어나는 현상으로 잘 알려져 있다. 또한 apoptosis 중 DNA 의 fragmentation 은 DNA ladder 를 통해 확인되었으며, 세포주기의 분포 변화 관찰실험을 통해 이러한 apoptosis 가 세포의 G0/G1 기에서 arrest 함으로써 일어나는 것을 확인할 수 있었다. 결론적으로, 본 실험을 통해 한국산 도라지에서 항산화와 항암효과를 가지는 생리활성물질이 각각 검색, 분리되었는데, 분리된 페놀성 wax ester 는 도라지에서 처음으로 발견됨과 동시에 그 항산화 활성이 처음으로 보고되었고, 도라지에서 분리된 polyacetylene 은 여러 종류의 암세포에서 성장을 억제시키는데, 특히 인체 결장암세포인 HCT-116 에서는 apoptosis를 유도하는 것을 확인하였다. In this study, the petroleum ether extracts from the root of Platycodon grandiflorum A. De Candolle (balloonflower), a plant used as traditional oriental medicine and also as a food material in Korea, were investigated on their antioxidant and anticancer activities. Crude petroleum ether extracts from dried P. grandiflorum root were fractionated into FI ∼ FV by a silica gel column chromatography with gradient solvents (petroleum ether : ethyl ether, 9:1∼5:5, v/v). First, the antioxidant activity of the fractions was evaluated based on lipid peroxidation inhibition and free radical scavenging activity. Among the fractions, FII (8:2) showed the highest antioxidant activity, and its antioxidant activity was closely related with the phenolic content in the fraction. The FII was further separated into 5 sub-fractions, FII-A∼E, by a preparative thin layer chromatography (TLC), and then the sub-fractions were screened for the antioxidant activity, which was comparable to that of commercial antioxidants such as α-tocopherol and BHA. The anticancer activity of FI ∼ FV was evaluated by MTT assay using human cancer cell lines (HT-29, HRT-18 and HepG2), and FIII(7:3) showed the highest growth inhibition effect on all cancer cell lines tested. The FIII was also sub-fractionated into FIII-A∼D by preparative TLC, and among the sub-fractions, FIII-B exhibited the highest and concentration-dependent cytotoxicity. From these results of activity screening, the potential as a source of highly active antioxidant and anticancer agents was confirmed in the petroleum ether extracts of P. grandiflorum root, and accordingly, the extract was further investigated for separation and evaluation of active compounds. For more specialized study on antioxidant, FII (8:2) that had the highest antioxidant activity was subjected to 2nd silica gel column chromatography using more detailed solvent gradients (petroleum ether : ethyl ether, 10:0∼8:2), and six sub-fractions FII-1∼6 were obtained. Then, the most active sub-fraction, FII-3, was separated again by HPLC to obtain two pure antioxidant compounds, Compounds 1 and 2. From the analysis by UV spectra, FAB-MS, IR, 13C and 1H NMR, both compounds were identified as wax esters that had the similar structures consisting of phenolic alcohol and fatty acid. Both had coniferyl alcohol as phenolic alcohol, but Compound 1 (M.W. 418) contained palmtic acid as fatty acid, whereas Compound 2 (M.W. 444) had oleic acid. The antioxidant activity, based on DPPH radical scavenging activity, was higher in Compound 2 than in Compound 1, which was also higher than that of BHA. In superoxide and nitric oxide radical scavenging tests, both compounds also exhibited higher activity than BHA and BHT. These wax ester compounds mostly have been identified in cuticle or suberin of vascular plants, but their function or activity, especially related with antioxidant activity, have been rarely studied. In this study, two wax ester compounds were newly identified in P. grandiflorum, and their antioxidant activity was also firstly reported. For the study of anticancer activity of P. grandiflorum extracts, FIII (7:3) showing the highest activity was also separated again into six sub-fractions FIII- 1∼6 using preparative TLC and subsequent four fractions FIII-2a∼d from FIII-2 using HPLC, and one active compound was obtained from active FIII-2c fraction. Although additional structural analysis was not performed, its basic structure was identified as a polyacetylene compound based on its typical UV absorbance spectrum. By MTT assay, this compound showed a significant growth inhibiting effect on human colon carcinoma cell, HCT-116, in time and concentration dependent manners at conditions tested. Especially at a concentration of 60 μg/mL, the compound showed over 50 % of cytotoxicity on cancer cells within 24 h, and after 72 h, almost 90 % of cancer cells were induced to cell death. For the study of this cytotoxicity in relation to apoptosis, a specific form of programmed cell death, the characteristic changes on cell morphology and DNA that commonly occur during apoptosis were investigated. Cell shrinkage and reduced cell number were induced by incubating HCT-116 with the compound, and cell nuclear fragmentation and apoptotic body formation were also observed after acridine orange / ethidium bromide staining, which are all typical morphological changes during apoptosis. DNA fragmentation during apoptosis was also confirmed by observing DNA ladder using electrophoresis. From all those evidences, it is concluded that this compound induced apoptosis in HCT-116 human colon carcinoma cell, and from the flow cytometry study, the apoptosis occurred by the arrest on the G0/G1 phase of cell cycle. Conclusively through this study, two wax ester compounds exhibiting highly antioxidant activity were firstly separated from the petroleum ether extracts of P. grandiflorum root. Also one polyacetylene compound showing cytotoxicity in human colon carcinoma cell by inducing apoptosis was obtained from those P. grandiflorum extracts.

Hepatitis B Virus X Protein : Molecular Basis of Intracellular Instability

김정환 Graduate School of Life Sciences & Biotechnology K 2003 국내박사

Hepatitis B virus (HBV) X protein (HBx) plays an essential role in viral replication and in the development of hepatocellular carcinoma. HBx has the capability to transactivate the expression of all HBV proteins including the viral core protein HBc. Consistent with its regulatory role HBx is relatively unstable and is present at low levels in the cell. I report here that the level of HBx was significantly reduced by the co-expression of HBc in cultured human hepatoma cells, whereas the level of HBx mRNA was unaffected. The repression of HBx by HBc was relieved by treating cells with the proteasome inhibitor MG132, indicating that HBc acts by stimulating the proteasome-mediated degradation of HBx. The inhibitory effect of HBc was specific to HBx and did not affect other proteins, including p53, a known target of proteasome. Although no direct physical interaction between HBc and HBx could be demonstrated in this study, mutational analysis indicated that the C-terminal half of the HBc is responsible for its inhibitory effect. These results suggest that HBc functions as a novel regulator of the HBV life cycle and of hepatocellular carcinogenesis through the control of HBx level via an inhibitory feedback-type of mechanism. Furthermore, it was shown that HBx contains bipartite recognition signals for proteasome-mediated degradation in its middle and C-terminal parts. In addition, lysine residues that serve as ubiquitination target site were characterized. Through site-directed mutagenesis, it was shown that ubiquitination was not restricted to any of the 6 lysine residues and that all the 6 lysines can be ubiquitinated with diverse susceptibility. Among the 6 lysines, the 140th lysine residue was ubiquitinated with highest efficiency. This result indicates that the 140th lysine may act as the main ubiquitin acceptor in physiological condition. Interestingly, one mutant in which all the 6 lysines were substituted by alanine still served a proteasome substrate, although it was free from being ubiquitination. It is thought, therefore, that the degradation of HBx is achieved through both ubiquitin-dependent as well as ubiquitin-independent pathway.

Formation and characterization of biopolymer-coated nano delivery system

방성환 Graduate School of Life Sciences and Biotechnology 2011 국내박사

Chitosan-coated nano-liposomes containing insecticides were prepared by ultrasonic homogenization and a combined use of ultrasonic homogenization and electro-spraying. And phosphatidylcholine (pc), cholesterol and chitosan were used as main materials. The physicochemical properties of the resulting samples were examined and compared. In the research, nano-liposomes were constructed using different types and concentrations of chitosan to regulate the mean size and surface charge. As the chitosan concentration (0.1?0.5%, w/v) and the degree of deacetylation increased, surface charge also increased. The encapsulation efficiency and release profile were measured by gas chromatography. Encapsulation efficiency decreased slightly as chitosan concentration increased (0.1?0.5%, w/v). The two methods yielded similar values and tendencies, except for encapsulation efficiency that differed by an average of 15%. As the intrinsic surface charge or concentration of the coating material increased, the release period of the entrapped core material was extended. The results indicate that diverse preparation conditions could affect the physicochemical properties and release profile of the resulting nano carrier systems. Insect mortality test based on the percentage of insects killed showed that sustainability was significantly different between the raw material (etofenprox) and chitosan-coated nanocarrier containing etofenprox.

(A)role of TAZ in tumorigenesis of breast cancer

방민수 Graduate School of Life Sciences and Biotechnology 2010 국내석사

The Study of Mitochondria Mediated-Apoptosis : Mitochondria mediated apoptosis induced by selenium and oxidative stimuli

김태수 Graduate School of Life Sciences and Biotechnology 2003 국내박사

미토콘드리아는 세포 내 ATP 에너지 대사뿐만 아니라 세포 사멸 기전에 중요한 역할을 담당한다. 미토콘드리아가 관여하는 세포 사멸 인자들은 대표적으로 활성화 산소종와 Bcl-2 family등을 들 수 있다. 본 연구는 셀레늄과 세포내에서 자발적으로 생성되는 활성화 산소종에 의한 세포 사멸 기전에 미토콘드리의 역할에 관한 내용을 연구하였다. 셀레늄은 생체 내에 있어서 반드시 필요한 미량원소로 작용한다. 최근에는 다양한 셀레늄 화합물을 사용하여 암세포를 사멸시키는 치료제로 사용할 수 있다는 가능성이 제시되고 있다. 본 연구를 통하여 셀레늄이 미토콘드리아의 막 단백질에 thiol 기를 산화시켜 MPT를 유도되고 이에 따라 선택적 투과성의 파괴를 통해 시토크롬 c가 세포질로 방출됨으로서 일련에 세포사멸 과정이 진행된다는 사실을 밝혔다. 또한 단백질 thiol 기의 산화에 의한 경로뿐 만 아니라 글루타치온과 셀레늄의 반응을 통해 생성되는 슈퍼옥사이드가 미토콘드리아의 MPT를 유도한다는 사실을 밝혔다. 미토콘드리아는 세포 내에서 가장 많은 활성화 산소종이 생성되는 기관이다. 이 같이 자발적으로 생성되는 활성화 산소종 생성이 당대사 경로에 따라 조절될 수 있고 이 결과 산화적 스트레스에 대한 저항성도 변화될 수 있다는 사실을 연구하였다. 이를 증명하고자 당대사과정을 변형시켜 미토콘드리아로 유입되는 환원력의 양이 각기 다른 두 세포주를 형질 변형을 통해 다음과 같이 구축하였다. Chinese Hamster Ovary (CHO) 세포에 cytosolic glycerol dehydrogenase (cGPDH)를 과량 발현시켜 미토콘드리아로 유입되는 환원력을 감소시킬 수 있는 세포주(CHO/cGPDH)와 anti-sense mRNA 기법을 사용하여 lactate dehydrogenase 합성을 억제 시켜 미토콘드리아로 유입되는 환원력을 증가시킨 세포주(CHO/anti-LDH)를 구축하였다. 그 결과 미토콘드리아로 유입되는 환원력에 따라 세포내 활성화 산소종의 양이 변화한다는 사실을 관찰하였고 산화적 스트레스에 대한 민감성 변화도 관찰할 수 있었다. 그리고 이들 세포주에 산화적 스트레스를 가하였을 때 세포내 활성화 산소종이 가장 많이 생성되는 세포주인 CHO/anti-LDH에서 세포 사멸 현상을 관찰 할 洙있었고 이때 미토콘드리아가 중요한 역할을 담당한다는 사실을 밝혔다. Mitochondria play a pivotal role in the induction of apoptosis in many cell types. Selenium is an essential trace element in mammals and is thought to play a chemopreventive role in human cancer, possibly by inducing tumor cell apoptosis. The effects of selenite on mitochondrial function were therefore investigated. Selenite-induced the oxidation and cross-linking of protein thiol groups, mitochondrial permeability transition (MPT), a decrease in the mitochondrial membrane potential, and the release of cytochrome c in mitochondria isolated from rat liver. These results thus indicate that selenite induces the MPT as a result of direct modification of protein thiol groups, resulting in the release of cytochrome c and a loss of mitochondrial membrane potential. Furthermore, selenite generated the superoxide (O_(2)^(●-)) on reaction with glutathione in the presence of mitochondria, and an O_(2)^(●-) scavenger inhibited their induction of the MPT. These results suggest that the pro-apoptotic action of selenium compounds is mediated by both thiol oxidation and the generation of O_(2)^(●-), both of which contribute to opening of the MPT pore. I hypothesized that sensitivity to oxidative stress in the cell could. be regulated by the alteration of respiratory substrate influx. To investigate the effect of the alteration of metabolic pathway on cell damage by oxidative stress, we developed dihydroforate reductase (dhfr)-deficient Chinese Hamster Ovary (CHO dhfr^(-)) cell through the over-expression of cytosolic glycerol 3-phosphate dehydrogenase (cGPDH, CHO/cGPDH) or a reduction of lactate dehydrogenase (LDH) using the anti-sense mRNA expression (CHO/anti-LDH). They have the increased metabolic pathway via oxidative phosphorylation in the order of CHO/anti-LDH, CHO dhfr^(-), and CHO/cGPDH. When three cell lines with different metabolic efflux were treated with oxidant, tert-butyl hydroperoxide (t-BOOH), the increase of flow rate through electron transport chain was more sensitive to oxidative stress. Taken together, we demonstrate that the alteration of efflux of electron transport chain via mitochondria plays an important role in aspects of regulation of oxidoresistance.

Characterization of genesrelated to cell size growth and CCN family in mouseovarian early folliculogenesis

김경화 Graduate School of Life Science and Biotechnology, 2006 국내석사

Identification and Characterization of Two Genes, Ferredoxin and ERD15, for Putative Interactors of Tsip1 (Tsi1- Interacting Protein 1) Protein : Tsip1 단백질과 상호 작용하는 식물 인자들에 대한 연구

이인주 Graduate School of Life Sciences and Biotechnology 2003 국내석사

Tsip1은 EREBP/AP2 전사 단백질인 Tsi1 과 상호작용하는 단백질로 분리되어졌으며 이런 Tsip1-Tsi1 의 상호작용은 Tsi1 유전자 발현을 조절하는데 특정 역할을 할 것이라고 여겨졌다. 이 논문에서는 Tsi1 단백질의 기능을 좀 더 구체적으로 규명하자고자 GAL4 yeast two hybid system을 이용하여 Tsip1 단백질과 상호작용하는 담배 인자들을 분리하였다. 먼저 dot-blot assay를 통하여 분리되어진 clone들을 발현 정도를 조사하였고, 이들 중 두 가지 유전자를 선발하여 연구하였다. 한 유전자는 담배의 ferredoxin으로 Tsip1과 상호작용하는 것이 확인되었고 다른 종내에 ferredoxin 단백질들과 유사한 형태를 보였다. ferredoxin은 뿌리를 제외한 모든 식물 기관에서 발현 되였으며 염에 의해서만 유전자의 발현이 증가되는 다른 ERD15 유전자 에서도 동일하게 나타났다. ERD15은 염 스트레스 외에도 살리실 산, TMV 감염에 의해서도 유전자의 발현이 증가 되였다. ferredoxin은 담배 내에서 여러 copy로 존재하였고 엽록체 내에 위치한다는 것을 확인하였다. 또한 Tsip1과 동시에 공동전이를 시켰을 때 두 단백질이 엽록체 내에 함께 존재하는 것을 관찰하였다. 식물체 내에서 ferredoxin 유전자의 발현은 억제시켰을 때는 잎맥을 따라 탈색되어지는 현상이 나타났다. 이런 결과들은 ferredoxin과 Tsip1 단백질이 Tsip1 단백질과 상호작용하는 식물인자임을 확인시켜 주며 이들이 식물의 저항성 반응에서 특정 기능을 수행하는 것을 암시해 준다. Tsip1 (Tsi1-intera ting protein 1), a novel DnaJ-type Zn finger protein was isolated as an interaction factor of Tsi1 which encodes EREBP/AP2-type transcription factor. To further determine the function of Tsip1, a tobacco cDNA library expressing the GAL4 activation domain fusion protein was screened using Tsip1 as a bait. Among the clones, two clones were selected for futher study. One gene was a tobacco ferredoxin whose translation product apparently interacted with Tsip1. Consistent with this observation, baterially expressed Tsip1 protein bound tightly to Ferredoxin in vitro. The deduced amino acid sequence showed a high degree of homology with ferredoxin proteins from other organisms. Southern blot analysis of genomic DNA revealed that ferredoxin belonged to a multigene family. The expression of ferredoxin gene was induced in response to salt treatment whereas other treatments such as pathogen, SA, ethephon or MV did not induce the expression of ferredoxin gene Subcellular localization of ferredoxin was examined by introducing ferredoxin::smGFP fusion construct into Arabidopsis protoplasts Ferredoxin protein was targeted to the chloroplast and co-localized with Tsip1 in the chloroplast. Virus-indued gene silencing of ferredoxin esulted in the phenotype of bleached leaves The other gene, ERD15 mRNA was induced by salt, SA treatments and TMV inoculation. Southern blot analysis showed that a small family of ERD15 was present in tobacco genome. The collected results suggest that ferredoxin, ERD15an be a putative interactor of Tsip1 and they can be involved in the plant defense response.

Identification of a Novel Protein D3UPCA from Halobacterium salinarium and Prediction of its Function

이문섭 Graduate School of Life Sciences and Biotechnology 2003 국내석사

Halobacterium salinarium is an extremely halophilic archaea which is able to live in the high salt environment. In a recent study, several halophilic archaea were found to have abilities to biodegradate organic hydrocarbon pollutants, but protein informations regarding to hydrocarbon degradation or tolerance in halophilic archaea have been known rarely. In this study, the protein expression profile of H. salinarium cultured under different diesel concentrations (0%, 2% and 4%) was investigated with the two-dimensional gel electrophoresis (2-DE). Proteins which show increasing of expression level in diesel media were identified using the MALDI-TOF and ESI-MS/MS analysis. Among them, a protein in spot 3 (named as ‘D3UPCA’) was selected which was up-regulated about 9 fold and found to have COG3388, uncharacterized protein conserved in archaea. The D3UPCA coding gene (named as ‘d3upca’) was cloned and expressed in E. coli, and purified by GST-fusion method. It’s function was predicted using various bioinformatics tools. Further characterization of this protein may reveal the possibility to make an industrial enzyme useful in developing organic stress-tolerant plants and animals.

Testis specific deubiquitinating enzyme Usp19 interacts with heat-shock protein 90

성민우 Graduate School of Life Science and Biotechnology, 2006 국내석사

Kinetics and in vivo fate of gene delivered in bone marrow-derived mesenchymal progenitor cells

정영신 Graduate School of Life Science and Biotechnology, 2005 국내석사