Effect of a Bombyx mori Protein Disulfide Isomerase on Production of Recombinant Antibacterial Peptides

구태원,김성완,최광호,김성률,강석우,박승원,윤은영 한국잠사학회 2013 International Journal of Industrial Entomology Vol.26 No.2

The insect baculovirus expression vector system (BEVS) is useful for producing biologically active recombinant proteins. However, the overexpression of heterologous proteins using this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we developed a versatile baculovirus expression and secretion system using Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion was found to improve the secretions and antibacterial activities of recombinant nuecin and enbocin proteins. Thus, we conclude that bPDI gene fusion is a useful addition to BEVS for the large-scale production of bioactive recombinant proteins.

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

ER-Golgi 분비 경로를 통해서 정확한 구조를 가지면서 post-translational modification 과정을 거친 재조합 단백질의 발현을 최대화하는 것은 ER stress반응에 대한 연구의 중요한 계기가 된다. 세포가 스트레스를 받지 않는 상태라도 ER stress signaling은 재조합 단백질의 생산량을 제한하고 품질을 떨어뜨리는 여러 가지 조건을 만들게 된다. ER stress signaling을 막는 여러 가지 방법들이 제시되고 있으며 표 2는 이러한 방법들 중 일부를 나타내고 있다. 일반적으로는 pro-survival 경로에 관련되어 있는 인자를 촉진하고 pro-apoptosis에 관련되어 있는 인자를 억제하는 것들이다. 그러나 ER stress 반응은 매우 복잡하고 적응과 사멸 기작(adaptation and elimination mechanism)의 중간 역할을 하기 때문에 ER stress에 관련된 주요 인자를 산업적으로 응용하기 위해선 이들의 기능에 대해 보다 깊은 연구가 이루어져야 한다. 현재까지 재조합단백질의 생산량을 최대한으로 높이는 방법은 ER stress 반응이 생기지 않도록 fed-batch process를 개선하고 세포 사멸 기작을 조절하며 단백질의 glycosylation 처리를 하는 것이다. 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.

누에 배양세포(Bm5)로부터 분리한 새로운 전사제어인자 ATFC의 특성분석

구태원,윤은영,김성완,최광호,황재삼,박수정,권오유,강석우 한국생명과학회 2003 생명과학회지 Vol.13 No.5

누에배양세포주(Bm5)에 N-glycosylation 저해제인 tunicamycin를 처리하여 인위적으로 UPR를 유도하고 이로부터 cDNA 유전자은행을 제작한 후, 정상세포주에 비하여 발현량이 증가하는 40개의 차별화 발현 cDNA 클론을 선발하였다. 차별화발현 클론 중에서 기존에 밝혀진 전사제어인자와 1차 아미노산의 구조적 유사성을 나타내는 클론(ATFC)에 대하여 유전자의 구조와 발현특성을 분석한 결과는 다음과 같다. 유전자의 구조분석 결과, ATFC는 효모의 전사제어인자 Hac1p와 구조적으로 매우 유사하게 $\alpha$-helix 상의 7개 아미노산 잔기 마다 leucine이 7회 반복하여 출현하는 leucine zipper 모티프가 존재하고 있었으며, leucine zipper 바로 앞쪽에는 분자 샤페론이나 folding enzyme의 프로모터 부위에 존재하는 UPRE에 결합할 것으로 추정되는 염기성 아미노산이 풍부한 basic region이 존재하고 있었다. 또한, ATFC 유전자에 대하여 분자샤페론 및 folding enzyme의 전사 촉진 기능을 해석하기 위하여 누에 배양세포주(Bm5)에 각종 스트레스 유도제를 처리한 후 ATFC의 발현특성을 분석한 결과, 정상 세포주에서는 발현이 되지 않았으나 스트레스 유도제가 처리된 세포주에서는 ATFC의 전사체가 강하게 발현됨을 확인 할 수 있었다. 따라서 ATFC 유전자는 소포체 내의 정확하게 접혀지지 않았거나 조립되지 못한 단백질을 정확하게 접혀지게 하고 조립되게 하여 정상구조를 가지는 단백질로 재생하는 분자샤페론이나 folding enzyme의 전사를 촉진시키는 효모 Hac1p와 매우 유사한 기능을 수행할 것으로 추정할 수 있었다. 이상의 결과는, 효모를 제외한 모든 생물종에서 UPR pathway에 관련한 전사제어인자의 최초의 보고이다. 억제로 야기되는 것 같다.증진시키기 위해 행동변화단계에 따른 맞춤형 교육 프로그램을 개발하여 적용하였고, 그 결과, 행동변화단계별 교육 프로그램이 자궁경부암 조기검진의 수검 행동을 증진시키는데 효과적인 것으로 나타났다.lomus thermophilum, Thermotoga neapolitana 등에서 밝혀진 바와 같이 glutamic acid 부위가 xylanase의 활성부위라 여겨진다.倍), 수층(水層)이 약(約) 100배(100倍)로 나타나 홍삼(紅蔘)엑기스의 갈변색소형성(褐變色素形成)은 비효소적(非酵素的) 갈변반응(褐變反應)인 amino-carbonyl 반응(反應)이 주도적(主導的) 역할(役割)을 하고 있음을 알 수 있다. (6) 총당(總糖)과 갈변반응속도(褐變反應速度)는 유의성(有意性)이 있었으며 $100^{\circ}C$의 경우 20시간(20時間)에 가장 색도(色度)가 높아 갈변반응속도(褐變反應速度)가 0.2로 나타났다. the esophageal mucous cells pf Bryzoichthys lysimus contained small amount of neutral mucin, while on the other hand a feww mucous cells contained small amount of neutral mucin and minimal amount of sialomucin. But the esophageal mucous cells of Takifugu pardalis contained considerable amount of neutral mucin only.분해가 더욱 촉진되었으며, 30℃에서 교반 처리를 행한 경우가 10℃에서 교반 처리를 행한 경우 보다 지방분해가 더욱 촉진되었다. 산양유 원유는 30℃에서 교반 처리 시간이 연장되어도 지방분해는 뚜렷한 증가를 나타내지 않았다.와 Cells respond to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) by increasing transcription of genes encoding molecular chaperones and folding enzymes. The information is transmitted from the ER lumen to the nucleus by intracellular signaling pathway, called the unfolded protein response (UPR). To obtain genes related to UPR from B. mori, the cDNA library was constructed with mRNA isolated from Bm5 cell lines in which N-glycosylation was inhibited by tunicamycin treatment. From the cDNA library, we selected 40 clones that differentially expressed when cells were treated with tunicamycin. Among these clones, we have isolated ATFC gene showing similarity with Hac1p, encoding a bZIP transcription factor of 5. cerevisiae. Basic-leucine zipper (bZIP) domain in amino acid sequences of ATFC shared homology with yeast Hac1p. Also, ATFC is up-regulated by accumulation of unfolded proteins in the ER through the treatment of ER stress drugs. Therefore we suggest that ATFC represents a major component of the putative transcription factor responsible for the UPR leading to the induction of ER-localized stress proteins.

Expression of the cyan fluorescent protein in fibroin H-chain of transgenic silkworm

구태원,최광호,김성렬,박승원,김성완 한국잠사학회 2017 International Journal of Industrial Entomology Vol.34 No.1

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누에 배양세포로부터 분리한 Protein Disulfide Isomerase 유전자의 발현 특성

구태원,윤은영,황재삼,강석우,권오유 한국생명과학회 2001 생명과학회지 Vol.11 No.5

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. We have isolated a cDNA encoding protein disulfide isomerase from Bombyx mori(bPDI). It has been characterized under ER stress conditions (dominantly induced by calcium ionophore A23187, tunicamycin and DTT), which is known to cause an accumulation of unfolded proteins in the ER. Furthermore, It has also been examined for tissue distribution(pronounced at the fat body), hormonal regulation (juvenile hormone, insulin and juvenile +transferrin; however, it is not effected by transferrin alone), and the effect of exogenous bacteria (peak at 16 h after infection) on the bPDI mRNA expression. The results suggest that bPDI is a member of the ER stress protein group, and it may play an important role in exogenous bacterial infection in fat body, and that homones regulate its expression.

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)의 정확한 병리적 기전은 아직 완전히 알려지지 못했지만 유전적 혹은 환경적요인인 깊이 관여하는 것으로 알려져 있다. 최근엔 IgA의 구조 이상이나 과다한 IgA가 생산되어 사구체에 침착되어 병변이 일어나는 것이 보고되고 있다. Megsin은 glomerular mesangium에서 지배적으로 발현되며 IgAN에서 과발현된다. Megsin의 생물학적 기능을 이해하기위하여 인간형 megsin이 과발현하는 D. melanogaster 형질전환체(actin-gal4>UAS-Megsin fly)를 만들었다. 이 형질전환체의 유전적 표현형은 melanin deficiency-abdomen이며 도입된 유전자와 단백질의 발현은 각각 RT-PCR과 Western blotting을 로 확인되었으며 megsin 유전자는 안정적으로 자손에게 유전되었다. 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.

Baculovirus Expression and Biochemical Characterization of the Bombyx mori Protein Disulfide Isomerase (bPDI)

구태원,윤은영,강석우,황재삼,권오유,김성완,최광호 한국잠사학회 2003 International Journal of Industrial Entomology Vol.7 No.2

멧누에(Bombyx mandarina)로부터 Chitinase를 코딩하는 cDNA의 분리 및 염기서열 결정

구태원,황재삼,성규병,윤은영,방혜선,권오유 한국생명과학회 1999 생명과학회지 Vol.9 No.4

Insects use chitinolytic enzyme to digest chitin in the exoskelton during the molting process. We have isolated and sequenced a chitinase-encoding cDNA from the silkworm, Bombyx mandarina, compared its sequenced with genes encoding chitinolytic enzymes from other sources. The insert DNA in the clone is 2,675 nucleotides long with an open reading frame of 1,695 uncletides that encodes a protein of 565 amino acids with a molecuar weight of 63.4 kDa. The 3' -untranslated region of 889 nucleotides is AT-rich and contains two putative polyadenylation signals. The N-terminal sequence of the encoded protein contains numerous hydrophobic residues characteristic of a leader peptide. The amino acid alignment revealed that the endo-$\beta$-N-acetylglucosaminidase had 83% and 97% homology with M. sexta and B. mori, respectively. The deduced amino acid had two highly conserved region at the amino acid residues 97-111 and 139-148 that were related to the existing chitinase.

A Powerful Ubiquitous Activity of Bombyx mori Heat Shock Protein 70 Promoter

구태원,김성완,Yong-Bo Kim,Seong-Ryul Kim,박승원,강석우,권오유,윤은영 한국유전학회 2011 Genes & Genomics Vol.33 No.6

For stable germline transformation, the promoter of Bombyx mori cytoplasmic actin gene (BmA3) has been used for ubiquitous expression of transgenes. So far, no strong promoter is available for ubiquitous expression in B. mori, excluding BmA3 promoter. To identify more powerful promoter than previously reported BmA3 promoter, we isolated 9 clones that show stronger signal compared to BmA3 by a dot blot hybridization. Among these 9 clones, we focused on one clone which has high amino acid homology (94%) with heat shock protein 70 gene of Trichoplusia ni. This clone, named bHsp70was ubiquitously expressed in all tissues and developmental stage of 5th instar B. mori larvae, and stimulated by thermal and ER stress. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-1003/+147) in the 5’-flanking region of bHsp70 gene,which has about 264 fold more intensive promoter activity than BmA3 promoter. Transcription activity of bHsp70 promoter under heat shock condition (42°C, 4 hr) was increased over 2 fold than normal condition. In bHsp70 promoter, there are an ER stress response element (ERSE) and a heat shock element (HSE). Accordingly, we suppose that the bHsp70promoter may be used for conditional expression of heterogonous proteins under thermal or ER stress. Moreover, the bHsp70 promoter was normally regulated in Bm5, Sf9, and S2 cells. Therefore, we suggest that bHsp70 promoter may be used more powerful and effectively for transgene expression in various insects containing B. mori as a universal promoter.

뽕나무 묘목생산을 위한 신소삽목 조건 구명

구태원,성규병,김호락,류근섭 한국잠사학회 1997 한국잠사곤충학회지 Vol.39 No.2

Most of the mulberry saplings in Korea have been produced by grafting. But the production of mulberry sapling by this method is very complex process and requires high level of technique and two years to produce saplings. Therefore, to develop the mulberry sapling production system by softwood cutting method which is suitable for mass production, several conditions on rooting and growth of cuttings were investigated. The rooting ability of cuttings varied according to mulberry varieties, showing the highest rooting ratio of 93.3% in cultivar of Shingwangppong. Of different soil texture as for rooting media, the clay was found to be the best; other media decreased in order as fallows; sand, sand loam and vermiculite. The shading ratio by 70% with polyethylene film showed the highest rooting ratio of the cuttings. Optimum day of cutting was around 50th day after sprouting, whereas the ratio of stem thicker than 7mm at the base of saplings in diameter was highest at 44th day.