재조합 단백질 생산을 위한 소포체 신호전달

구태원,윤은영,강석우,권기상,권오유,Goo, Tae-Won,Yun, Eun-Young,Kang, Seok-Woo,Kwon, Ki-Sang,Kwon, O-Yu 한국생명과학회 2007 생명과학회지 Vol.17 No.6

The endoplasmic reticulum (ER) is an important intracellular organelle for folding and maturation of newly synthesized transmembrane and secretory proteins. The ER provides stringent quality control systems to ensure that only correctly folded proteins exit the ER and unfolded or misfolded proteins are retained and ultimately degraded. The ER has evolved stress response both signaling pathways the unfolded protein response (UPR) to cope with the accmulation of unfolded or misfolded proteins and ER overload response (EOR). Accumulating evidence suggests that, in addition to responsibility for protein processing, ER is also an important signaling compartment and a sensor of cellular stress. In this respect, production of bio-functional recombinant-proteins requires efficient functioning of the ER secretory pathway in host cells. This review briefly summarizes our understanding of the ER signaling developed in the recent years to help of the secretion capacities of recombinant cells.

인간 신장질환 유발인자가 발현하는 형질전환 초파리 구축

구태원,권기상,권오유,Goo, Tae-Won,Kwon, Ki-Sang,Kwon, O-Yu 한국생명과학회 2007 생명과학회지 Vol.17 No.5

IgA nephropathy(IgAN) is considered to be a multifactorial disease with genetic and environmental factors contributing to its pathogenesis. The genes involved in susceptibility and progression of the disease have not yet been clearly elucidated. Megsin is an important candidate gene, predominantly expressed in glomerular mesangium and upregulated in IgAN. To understand biological function of megsin, in this work we have produced transgenic D. melanogaster fly over-expressing human megsin(actin-gal4>UAS-Megsin fly). Introduced human megsin was confirmed by RT-PCR and Western blotting, respectively. Its phenotype is melanin deficiency-abdomen and the megsin gene is stably transferred to the next generations.

소포체 스트레스에 대한 Protein Disulfide Isomerase의 세포보호효과

구태원,윤은영,김성완,최광호,강석우,권기상,권오유,Goo, Tae-Won,Yun, Eun-Young,Kim, Sung-Wan,Choi, Kwang-Ho,Kang, Seok-Woo,Kwon, Ki-Sang,Kwon, O-Yu 한국생명과학회 2007 생명과학회지 Vol.17 No.8

In the previous our study, a cDNA that encodes protein disulfide isomerase from Bombyx mori (bPDI)was isolated and characterized. bPDI has an open reading frame of 494 amino acids contained two PDI-typical thioredoxin active site of WCGHCK and ER (endoplasmic reticulum) retention signal of the KDEL motif at its C-terminal. Recent studies have demonstrated that misfolded proteins are accumulated in many diseases including Alzheimer’s, goiter, emphysema, and prion infections. bPDI was over-expressed or knock-downed in Sf9 cells to study the relationship between bPDI expression and protections against protein misfolding. bPDI gene was cloned in insect expression vector pIZT/V5-His for over-expression and bPDI double-stranded RNA (dsRNA) was generated for knock-down. Over-expression of bPDI significantly improved survival rate, but bPDI dsRNA transfection significantly reduced survival rate after 48 hours exposure. In mock-transfected or wild-type cells had no significant effect. The results support the view that bPDI is one of the important intracellular components for cell protect mechanism, especially, against ER stress such as protein misfolding.

누에로부터 핵다각체병 바이러스 방어관련 유전자 정보 분석

구태원 ( Tae Won Goo ),홍선미 ( Sun Mee Hong ),김성완 ( Sung Wan Kim ),최광호 ( Kwang Ho Choi ),김성렬 ( Seong Ryul Kim ),박승원 ( Seung Won Park ),강석우 ( Seok Woo Kang ),윤은영 ( Eun Young Yun ) 한국잠사학회 2012 한국잠사곤충학회지 Vol.50 No.2

Silkworm larvae often suffer from viral infections causing heavy losses to the economy of silk industry. Insects exhibit both humoral and cellular immune responses that are effective against various pathohens like bacteria, fungi, protozoa, etc., but no insect immune responses is effective against viral infection. To obtain genes related to insect antiviral immunity from Bombyx mori, the cDNA library was constructed from B. mori nucleopolyhedrovirus (BmNPV)-infected B. mori. From the cDNA library, we selected 411 differentially expressed clones, and the 5` ends of the inserts were sequenced to generate ESTs. In this work, 135 unigenes were generated after the assembly of 411 differentially expressed clones ESTs. Of these 135 unigenes, we selected 109 antiviral response-related candidates except 26 clones that high similarity with genes derived from BmNPV. Among 109 unigenes, a total of 80% had significant matches to genes from other organisms in the database, wheres 20% of the unigenes had not matched in the database. Functional groups of these sequences with matches in database were constructed according to their putative biological function. Three largest categories were control of cellular oraganization (52%), metabolism (20%), and protein fate (10%). The genetic information reported in this study will provide more information about antiviral-related genes in silkworms.

Tunicamycin 을 처리한 누에 배양세포 ( Bm5 ) 로부터 cDNA 유전자은행 제작 및 차별화 클론 선발

구태원(Tae Won Goo),윤은영(Eun Young Yun),황재삼(Jae Sam Hwang),강석우(Seok Woo Kang),이진성(Jin Sung Lee),박수정(Soo Jung Park),이광식(Kwang Sik Lee),권오유(O Yu Kwon) 한국유전학회 2001 Genes & Genomics Vol.23 No.2

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the transcriptional induction of molecular chaperones and folding enzymes localized in the ER. Thus, eukaryotic cells possess an intracellular signaling pathway from the ER to the nucleus, called the unfolded protein response (UPR) pathway. To obtain genes related to UPR from Bombyx mori Bm5 cell, the cDNA library was constructed with mRNA isolated from Bm5 cell line in which N-glycosylation was inhibited by tunicamycin treatment (5 ug/ml). From the cDNA library, we selected 40 clones that differentially expressed when cells treated with tunicamycin, and produced expressed sequence tags (ESTs). Among these clones, we have isolated TmInc329 clone showing high similarity with ATF (encodes a bZIP transcription factor) of M. muscululs. Basic-leucine zipper (bZIP) domain in amino acid sequences of TmInc329 shared homology with several transcription factors, yeast Hac1p, human CREB and mouse ATF. Also, TmInc329 clone is up-regulated when N-glycosylation of newly synthesized proteins in the ER is inhibited by tunicamycin treatment. Therefore we suggest that TmInc329 clone is a gene responding to accumulation of unfolded proteins in the ER.

모바일 환경에서 캐주얼 게임의 난이도에 따른 게임 이용 지속성에 관한 연구

구태원(Tae-Won Goo),김효남(Hyo-Nam Kim) 한국컴퓨터정보학회 2018 한국컴퓨터정보학회 학술발표논문집 Vol.26 No.1

최근 급속도로 성장하는 모바일 시장은 게임 산업에서 가장 대표적인 게임 플랫폼으로 국내외에서 상당히 많은 수익과 성장을 이루고 있다. 매년 새로운 게임이 출시되서 유저들이 많이 플레이하지만 정작 몇 주 만에 유저들이 많이 빠져나오면서 실패한 게임들이 매년 나온다는 것이다. 본 논문에서는 실패하는 게임 개발을 줄이고 모바일게임 장르중에 대표적인 캐주얼 게임을 대상으로 남이도 조절을 통해서 유저들이 몰입하는 게임을 개발할 수 있는 내용을 제안하고자 한다.

누에 배양세포 ( Bm5 ) 로부터 Protein Disulfide Isomerase 유전자 분리 및 특성

구태원(Tae Won Goo),윤은영(Eun Young Yun),황재삼(Jae Sam Hwang),강석우(Seok Woo Kang),박수정(Soo Jung Park),권오유(O Yu Kwon) 한국유전학회 2001 Genes & Genomics Vol.23 No.3

Many secreted proteins have disulfide bonds that are important for their structure and function. Protein disulfide isomerase (PDI, EC 5.3.1.4.), an enzyme that catalyzes the formation and rearrangement of thiol/disulfide exchange reactions, is a resident of the endoplasmic reticulum (ER). The subcellular localization and its function as catalyst of disulfide bond formation in the biosynthesis of secretory and cell membrane proteins suggest that PDI plays a key role in the secretory pathway. To obtain genes related to molecular chaperone and the ER foldase from the Bombyx mori Bm5 cell line, the cDNA library was constructed with mRNA isolated from Bm5 cell line treated with by tunicamycin (5 ug/ml). We have isolated a cDNA encoding protein disulfide isomerase (bPDI), which consists of an open reading frame of 484 amino acids (55.6 kDa). It shows PDI-typical two active thioredoxin sites of CGHC and on ER retention signal of KDEL motif at its C-terminal. The bPDI protein shared less than 55% of the amino acid sequence motif at its C-terminal. The bPDI protein shared less than 55% of the amino acid sequence homology with other reported PDIs. bPDI is most genetically similar to the D. melanogaster PDI.

Establishment of a Stable Cell Line Expressing Human BMP2/7-PTD for Efficient Osteogenic Induction

Seung-Won Park(박승원),Seok-Woo Kang(강석우),Tae-Won Goo(구태원),Seong Ryul Kim(김성렬),Soon-Young Paik(백순영) 한국생명과학회 2012 생명과학회지 Vol.22 No.4

유전자 재조합 이형이합체 인간 뼈 형성촉진인자(rhBMP)들은 뼈 재생을 위한 조직공학연구에 중요한 요소들이다. 그러나 실제 뼈 재생에 관한 연구를 수행함에 이들을 이용하는 것은 뼈 형성 촉진인자들이 짧은 반감기를 가지며 비교적 고농도의 단백질을 사용해야 하기 때문에 그만큼 많은 비용이 소요된다는 문제점을 가지고 있다. 이러한 한계를 뛰어넘기 위하여, 본 연구에서는 단백질 전달 서열(PTD)이 융합된 인간 뼈 형성촉진인자 2와 7이 이형이합체 유전자 재조합 단백질(rhBMP2/7-PTD)을 발현하는 세포주를 확립하였다. 이형이합체의 형성은 BMP 2와 BMP 7 단백질 및 PTD 영역의 사이에 각각 4개의 glycine 아미노산 염기서열이 첨가될 수 있도록 하여 각 단백질의 folding이 자유롭도록 디자인하였다. 이렇게 개발된 세포주는 고농도의 rhBMP2/7-PTD을 지속적으로 발현하여 배양액 내로 분비함으로 조직공학용 연구 및 개발에 효율적으로 이용 할 수 있다. 이상의 세포주에서 발현된 rhBMP2/7-PTD 단백질은 뼈 세포분화 유도를 확인 할 수 있는 ALP 활성을 나타냄으로써 뼈 성장촉진 단백질로서 생물학적인 활성을 가지고 있음을 보였다. 본 연구의 결과로 개발된 rhBMP2/7-PTD 형질전환 세포주는 향후 뼈 조직 재생과 같은 연구에 중요하고 효과적인 도구로 이용될 수 있을 것으로 사료된다. Heterodimeric recombinant human bone morphogenetic proteins (rhBMPs) are powerful tools for bone tissue engineering. However, BMPs have several important limitations in their application to bone regeneration. BMPs have a short half-life and must be used in high concentrations, which may be cost-inefficient. To overcome these problems, we established a stable cell line that expressed the fusion protein comprised of recombinant human BMP2/7 heterodimer protein and PTD (rhBMP2/7-PTD). This stable cell line enabled high process yields by continuously expressing rhBMP2/7-PTD products at high levels throughout cultivation. This synthesized BMP7 was fused to a BMP2 protein with four glycine residues (to allow free bond rotation of the domains) and PTD. To demonstrate that the rhBMP2/7-PTD protein that was secreted from an rhBMP2/7-PTD-expressing stable cell line exhibited biological activity consistent with its role as an osteogenic differentiation induction growth factor, we evaluated BMP-induced ALP activity. Our results suggest that this cell line may be a powerful and efficient tool for applications such as bone tissue regeneration.

갑상선자극호르몬에 의한 분자\ulcorner페론 ERp29 유전자의 발현

박수정,이웅희,구태원,윤은영,황재삼,김호,송민호,권오규,Park, Soo-Jung,Lee, Woong-Hee,Goo, Tae-Won,Yun, Eun-Young,Hwang, Jae-Sam,Kim, Ho,Shong, Min-Ho,Kwon, O-Yu 한국생명과학회 2000 생명과학회지 Vol.10 No.2

This experiment was performed to evaluate the effect of TSH (thyroid-stimulating) on the ERp29 (endoplasmic reticulum resident 29 kDa protein) gene expression in the rat thyrocytes of FRTL-5 cells. Although ERp29 mRNA was constantly expressed, its expression began to increase remarkably from 10-9 M TSH. and its maximum expression was at 5×10-9 M TSH (about 3.5 fold). On the other hand, the effect of TSH on the abundance of ERp29 mRNA started within 6 h, and peaked at 8 h (about 2.5 fold). Actinomycin D (transcription inhibitor) strongly blocked this effect while cycloheximide (translation inhibitor) did not. The half-life of ERp29 mRNA was about 4.5 h in the presence or absence of TSH that was not affected by the stability of ERp29 mRNA. The effect of TSH on the ERp29 gene expression was specific, while other growth factors (transfferin, insulin, and hydrocortisone) did not alter its expression. Our data indicate for the first time that the expression of ERp29 is regulated transcriptionally by TSH in the thyrocytes.

Characterization of the Promoter Controling the Stage-Specific Gene Expression of Bombyx mori

박승원(Seung-Won Park),최광호(Gwang-Ho Choi),구태원(Tae-Won Goo),김성렬(Seong Ryul Kim),강석우(Seok-Woo Kang) 한국생명과학회 2011 생명과학회지 Vol.21 No.10

본 연구에서는 누에의 초기 배아시기에 유전자 발현 조절이 가능한 EEG-704 promoter를 개발하고자 하였다. Promoter의 핵심 영역을 결정하기 위하여, 10개의 서로 다른 partial mutant clone들을 만들고 이를 Sf9 곤충세포주에 도입하여 luciferase assay 방법을 사용하여 각각의 clone의 활성을 분석하였다. Constitutive promoter인 BmA3 promoter에 의한 활성과 비교하였을 때, 약1.5 kb의 promoter 염기서열을 포함하는 clone이 가장 높은 luciferase 발현율을 나타내었다. 특히 EEG-704 유전자의 경우 BLAST를 이용한 유전자 비교·분석의 결과 누에의 열충격 단백질 20.8 (BmHsp20.8) 과 동일한 것으로 밝혀졌으며, 정상 온도조건과 비교하였을 때 열충격을 가한 조건하에서 발현율이 증가하는 현상을 나타내었다. 특이적으로 발생단계에서 직·간접적으로 발현 조절이 가능한 이러한 promoter는 여러 유용 재조합 단백질 생산을 위한 형질전환 누에 개발 시 매우 유용할 것으로 생각된다 We characterized embryo early gene (EEG)-704 promoter of the silkworm Bombyx mori, which is specifically regulated in the development stages. To determine core promoter region, 10 different partial mutant clones were tested by luciferase assay in Sf9 cells. About 1.5 kb promoter shows higher luciferase activity than constitutive promoter (BmA3). Interestingly, EEG-704 shares the same DNA sequences with BmHsp20.8 by the result of BLAST analysis; its expression is also increased under heat shock condition. Development of such promoter inducible, directly or indirectly in the developmental-stage, is very useful in making recombinant proteins in transgenic silkworms.