근소포체의 단백질 및 당단백질 조성에 관한 연구

박영철 한국통합생물학회 1990 동물학회지 Vol.33 No.2

토끼의 골격근에서 근소포체를 순수 분리하여 SDS-polyacrylamide gel전기영동법과 125 I-concanavalin A표지법으로 단백질과 당단백질의 조성을 조사하였다. 전기영동사에 나타난 대표적인 단백질은 $Ca^2$+-AThase, 80 Kd protein,calsequestrin,high affinity calcium binding protein, intrinsic glycoprotein이었으며, 160 Kd protein, 94 Kd protein,38 Kd protein, 34 Kd protein,24 Kd proteins도 존재하였다.특히, 막성계에 있는 heak protein으로 알려져 있는 80 Kd protein은 본 연구를 통해 주로 근소포체의 terminal cisternae에 들어 있음이 확인되었다. 한편 125 I-concanavalin A표지에 의해 전기영동성에 나타난 대표적인 당단백질은 160 Kd glycoprotein, 94 Kd glycoprotein, calsequestrin, intrinsic glycoprotein의 4종이었다. 이 가운데 94 Kd glycoprotein은 94 Kd glucose-regulated protein으로 추정되며, 본 연구를 통해 근소포체에서도 특히 T-tubule에 다량으로 존재함이 밝혀졌다. Sarcoplasmic reticulum subfractions were isolated from rabbit sarcoplasmic reticulum vesicles using ultracentrifugation in a continuous sucrose gradient (12.5% 50%) after French pressure treatment. And proteins in sarcoplasmic reticulum were detected by SDS-polyacrylamide gel electrophoresis and glycoproteins were identified through the reaction with 1251-concanavalin A.The electrophoresis showed that sarcoplasmic reticulum contained predominantly $Ca^2$+-AThase and calsequestrin along with high affinity calcium binding protein, intrinsic glycoprotein 160 Kd, 94 Kd, 80 Kd, 38 Kd, 34 Kd and 24 Kd proteins. Among these, the protein of about 80 Kd which has been known as one of heat shock proteins was especially enriched in the terminal cistemae of sarcoplasmic reticulum. Meanwhile, autoradiogram of 125 I-concanavalin A bound to the stained gels showed the distribution of glycoproteins which included 160 Kd glycoprotein, 94 Kd glycoprotein, calsequestrin and intrinsic glycoprotein Among these, the protein of about 160 Kd was especially enriched in longitudial sarcoplasmic reticulum and T-tubule, and the protein of about 94 Kd which has been known as one of glucose-regulated proteins was also enriched in T-tubule and sharply reduced in terminal cistemae.

Optimal Conditions for the Expression of Glycoprotein E2 of Classical Swine Fever Virus using Baculovirus in Insect Cells

배성민,이승희,곽원석,안용오,신태영,우수동 한국잠사학회 2014 International Journal of Industrial Entomology Vol.29 No.2

The structural proteins of classical swine fever virus (CSFV) consist of nucleocapsid protein C and envelope glycoprotein Erns (E0), E1 and E2. Among them, E2, the most immunogenicof the CSFV glycoproteins, induces a protective immune response in swine. In this study, todetermine the optimal expression conditions of glycoprotein E2 using baculovirus system,we investigated the influence of insect cells and media to the expression of recombinant E2. Recombinant virus containing glycoprotein E2 coding gene was constructed with bApGOZADNA. Expression of the glycoprotein E2 was analyzed by SDS-PAGE and Western blotanalysis using anti-CSFV E2 monoclonal antibodies. Expression of glycoprotein E2 in Sf21cells was first observed after 3 days and reached a maximum on the 5th day after infection. Furthermore, the highest levels of glycoprotein E2 expression were observed at multiplicity ofinfection (MOI) of 5. When three different insect cell lines (Sf21, High-Five and Se301) weretested, High-Five cells showed the highest production. In addition, four different serum-freeand serum-supplemented media, respectively, were tested for the expression of glycoproteinE2 and the budded virus (BV) titers. As a result, serum-supplemented medium provided thebest conditions for protein production and the BV yield.

Expression and Characterization of Human Immunodeficiency Virus Type 1 Envelope Mutant Glycoproteins by Using Baculovirus Expression System

Lee, Yoon,Ryu, Ji-Yoon,Lee, Kil-Soo,Bae, Yong-Soo,Choi, Soo-Young,Park, Jin-Seu The Korean Society for Microbiology 2002 Journal of Bacteriology and Virology Vol.32 No.4

Considerable effort has been directed at understanding the structure and function of HIV-1 envelope glycoproteins. It has been difficult to characterize HIV-1 envelope glycoproteins due to the limited availability of these proteins from virus particles or infected cells. To facilitate the structural and functional analysis of HIV-1 envelope glycoproteins, recombinant baculoviruses were generated to express wild type or mutant HIV-1 envelope glycoproteins. The gp160 precursor protein as well as the gp120 glycoprotein were detected in the cell infected with recombinant BacENVw.t containing wild type HIV-1 envelope gene. In the insect cells infected with recombinant BacENVc with mutations at the cleavage site of gp160, a precursor form of envelope glycoprotein was produced, but not secreted into the culture medium. However, the insect cells infected with recombinant BacENVc/t containing both mutations at the cleavage site and membrane spanning region produced mutant envelope glycoproteins that were efficiently secreted into the culture medium in the form of precursor. Therefore the recombinant HIV-1 envelope glycoproteins produced in this system would be useful as immunogens in the development of a vaccine against AIDS.

2차원적 전기영동에 의한 톡소포자층 당단백 항원의 분석

황일영,최인욱,신대환,이영하 충남대학교 의학연구소 2003 충남의대잡지 Vol.30 No.2

Serious disease due to Toxoplasma gondii occurs in congenitally infected children and in immunosuppressed patients, particularly in the case of HIV infection. Glycoproteins modulate the physicochemical properties, and are involved in biological and immunological activities. Although there are many reports about the immune responses after T. gondii infection, there are few reports to identify the glycoproteins from T. gondii. For this reason, we purified the glycoproteins from tachyzoites and cysts of T. gondi and then evaluated the antigenic characterizations of toxoplasmic glycoproteins. Tachyzoites of T. gonii were purified by Percoll-gradient centrifugation. Glucosamine was the major monosaccharides released from the tachyzoites of T. gondii by acid hydrolysis methods. In order to define the antigenicities of both strains, we did 2-dimensional electrophoresis(2-DE). Over 40 protein spots were reproducibly separated in tachyzoites of T. gondii by 2-DE in range of the pH 5-8. Glycoproteins were isolated from tachyzoites of T gondii by Con A-Sepharose 4B affinity chromatography. The major bands of glycoproteins were approximately 22 kDa, 30 kDa and 38 kDa in tachyzoites of T. gondii.

Investigation for Expression of the Porcine Pseudorabies Virus Glycoproteins in Bacteria and Insect Cells

Bit Na Rae Yun,Sung Min Bae,Jae Bang Choi,Jun Beom Lee,Hee Jung Kim,Yeon Ho Je,Byung Rae Jin,Soo Dong Woo 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.05

Aujeszky's disease (AD), also called pseudorabies, is an infectious viral disease caused by an alpha herpes virus and has domestic and wild pigs, as well as a wide range of domestic and wild animals, as the natural host. Aujeszky's disease virus (ADV) virions contain several envelope glycoproteins. Among them, glycoproteins gB, gC and gD are regarded as the major immunogenicity proteins and the antibodies induced by them can neutralize virus in vitro or in vivo. In this study, we investigated expression of these glycoproteins using the bacterial and baculovirus expressionn system. Successful expression of ADV glycoproteins in E. coli was confirmed by SDS-PAGE and Western blot analysis and their optimal expression condition was determined. However, the recombinant proteins generated in the bacterial expression system which lacks glycosylation process frequently lose their biological activity. We tried to express the ADV glycoproteins using the baculovirus expression vector system. The recombinant gB, gC and gD were detected at approximately 100, 60 and 50 kDa on SDS-PAGE and Western blotting, respectively. The optimal expression conditions were determined for MOI(multiplicity of infection) and post-infection days. One MOI and 4 or 5 days post-infection were the best conditions for the expression of the ADV glycoproteins in Sf21 cells. We are currently investigating the antigenicity of recombinant proteins using experimental animals.

뽕잎 당단백질의 항산화능과 Raw 264.7 세포에 있어서 bisphenol A에 유도된 신호전달인자의 억제

심재웅(Jae-Uoong Shim),이세중(Sei-Jung Lee),오필선(Phil-Sun Oh),임게택(Kye-Taek Lim) 한국식품과학회 2007 한국식품과학회지 Vol.39 No.2

본 연구에서는 뽕잎 당단백질의 활성을 알아보기 위하여 뽕잎 당단백질의 안정성 및 특성을 알아보고, hydroxyl 라디칼, super-oxide anion 라디칼, DPPH 라디칼 등의 활성 산소종에 대한 항산화 효과들 살펴보았다. 또한 Raw 264.7 세포에 환경호르몬의 일종인 BPA와 뽕잎 당단백질(32kDa)을 함께 처리 하여, 뽕잎 당단백질의 활성산소증과 NO의 소거 능력뿐만 아니라 염증 매개성 단백질들 [NF-κB(p50)와 iNOS]의 활성 역제능력 대하여 평가하였다. 뽕잎 당단백질은 금속이온에는 다소 약하지만 온도와 pH에는 안정적인 특징을 지니고 있었으며, 탁월한 hydroxyl 라디칼, superoxide anion 라디칼, DPPH 라디칼 소거 능력을 가지고 있었다. 이러한 항산화 능력을 지닌 뽕잎 당단백질을 BFA와 함께 Raw 264.7 세포에 처리한 결과, BPA만 처리된 Raw 264.7 세포의 활성 산소종 양은 8시간째에, 그리고 NO 양은 24시간째에 현저히 증가한데 반해, BPA와 함께 뽕잎 당단백질을 처리한 Raw 264.7 세포에서는 같은 시간 동안에 농도에 비례하여 활성 산소종 및 NO양이 유의적으로 감소한 것을 알 수 있었다. 또한 8시간 동안 BPA저리에 의해 활성화된 Raw 264.7 세포의 NF-κB (p50)와 iNOS 단백질들은 함께 처리한 뽕잎 당단백질의 농도에 비례하여 현저히 억제되었다. 따라서, 이러한 결과를 종합해 보면, 뽕잎 당단백질은 높은 안정성과 강력한 항산화 효과를 가지고 있었으며, 이러한 항산화 효과가 환경 호르몬(BPA)에 의한 활성산소종 및 NO 생성을 저해한 뿐만 아니라, NF-κB(p50)와 iNOS의 활성을 억제함으로써 Raw 264.7 세포의 염증 신호전달을 막는데 영향을 끼쳤을 것으로 생각 된다. The present study investigated anti-oxidative and anti-inflammatory activity of glycoprotein isolated from Morus Indica Linne (MIL glycoprotein). We found that MIL glycoprotein has a molecular weight of 32 KD and consists of carbohydrate (40.03%) and protein (59.97%), and that it has a strong scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical (OH), and superoxide anion (O₂ㆍ?) radicals. In addition, MIL glycoprotein had a stable character and an optimal DPPH radical scavenging activity in the alkaline and neutral pH solution, and up to at 105. However, the results indicated that it has a minimal scavenging activity in the metal ionic solution (Ca²?, Mn²?, and Mg²?) in the presence of EDTA. In addition, we further investigated whether MIL glycoprotein scavenges oxygen radicals and blocks inflammation-related signals in the bisphenol A (BPA)-stimulated Raw 264.7 cells. The results in this study showed that it has a character to scavenge the productions of reactive oxygen species (ROS) and nitric oxide (NO) dose-dependently. Also it blocked the activities of inflammation-related signals such as nuclear factor-kappa B (NF-κB) and inducible nitric oxide synthase (iNOS). For example, it had an inhibitory effect on the activation of NF-κB (p50) and iNOS proteins at 200 ㎍/㎖ MIL glycoprotein. Here, we speculate that MIL glycoprotein is one of natural antioxidants and of modulators of the BPA-induced inflammation.

Characterization of S2 Glycoprotein gene of Infectious Bronchitis Virus isolated in Korea

Won-Gu Jeong(정원구),Hae-Won Jung,Hyuk-Moo Kwon 한국가금학회 2007 한국가금학회 정기총회 및 학술발표회 Vol.24 No.-

본 연구에서는 한국에서 분리된 infectious bronchitis virus(IBV)의 spike(S) gene 중에서기 S2 glycoprotein gene의 염기와 아미노산 서열을 결정하여 기존의 분리주와 비교하였고 phylogenetic tree를 작성하였다. 외국 분리주의 경우 S1 glycoprotein gene을 기준으로 작성한 phylogenetic tree 혈청형에 따라 분류되고 S2 glycoprotein gene을 기준으로 작성한 phylogenetic tree도 S1에 의한 결과와 밀접한 연관성을 갖는 것으로 보고되었으나 국내분리주의 경우 S1 glycoprotein에 의한 phylogenetic tree가 혈청형에 따라 분류되었으나 S2 glycoprotein gene에 의한 phylogenetic tree의 경우 모든 혈청형의 IBV 분리주가 하나의 cluster로 분류되는 특성을 나타내었다. 본 실험결과 국내 IBV 분리주의 경우 S2 glycoprotein gene이 S1 gene과는 다르게 변이하고 있는 것으로 조사되었다.

연구보문 : 식물에서 인체와 유사한 N-glycan 구조를 가진 당단백질 생산에 대한 최근 연구동향

심준수 ( Joon Soo Sim ),신동진 ( Dong Jin Shin ),한범수 ( Bum Soo Hahn ) 한국국제농업개발학회 2011 韓國國際農業開發學會誌 Vol.23 No.3

Several host systems including mammalian cells, yeasts, plants, bacteria, insects and transgenic animals have been employed to produce recombinant therapeutic glycoproteins. Among them, plant system has many advantages such as the low cost, animal pathogens-free, eco-friendly production, and ease of scale up. However, plants are unable to perfectly synthesize human-type N-glycans of therapeutic glycoproteins. It could represent a severe limitation on the use of therapeutic glycoproteins produced from transgenic plants. In this article, we focus on the importance of N-glycosylation, the advantage of molecular farming and its progress strategies, the difference of N-glycan structure between animals and plants, the risk of potential immunogenicity of plant glycoproteins, the strategies to reduce the potential immunogenicity by N-glycosylation engineering including endoplasmic reticulum (ER) targeting of therapeutic glycoproteins, the inhibition of plant-specific glycosyltransferases, and the expression of human specific glycosyltransferases to synthesis galactosylated and sialylated N-glycans in plants.

식물에서 인체와 유사한 N-glycan 구조를 가진 당단백질 생산에 대한 최근 연구동향

심준수,신동진,한범수 한국국제농업개발학회 2011 韓國國際農業開發學會誌 Vol.23 No.3

Several host systems including mammalian cells, yeasts, plants, bacteria, insects and transgenic animals have been employed to produce recombinant therapeutic glycoproteins. Among them, plant system has many advantages such as the low cost, animal pathogens-free, eco-friendly production, and ease of scale up. However, plants are unable to perfectly synthesize human-type N-glycans of therapeutic glycoproteins. It could represent a severe limitation on the use of therapeutic glycoproteins produced from transgenic plants. In this article, we focus on the importance of N-glycosylation, the advantage of molecular farming and its progress strategies, the difference of N-glycan structure between animals and plants, the risk of potential immunogenicity of plant glycoproteins, the strategies to reduce the potential immunogenicity by N-glycosylation engineering including endoplasmic reticulum (ER) targeting of therapeutic glycoproteins, the inhibition of plant-specific glycosyltransferases, and the expression of human specific glycosyltransferases to synthesis galactosylated and sialylated N-glycans in plants. 1989년에 처음으로 형질전환식물체를 이용하여 재조합 항체가 생산된 이래로(Hiatt et al., 1989) 분자농업 및 관련 기술들은 매우 빠르게 발전해왔다. 식물 시스템을 이용하여 의료용 단백질 및 산업용 소재 등의 생산은 경제성 및 안전성 측면에서 동물세포 시스템 보다 우위에 있으며, 단점으로 남아있는 낮은 발현양, 재조합 당단백질 내 식물특이 당쇄구조의 존재로 인한 면역성 문제 및 동물 N-glycan 내 존재하는 당쇄구조의 결핍으로 인한 생체 내 안전성의 부족 등의 문제점들이 생명공학기술의 발달에 의해 현재 빠르게 극복해 나가고 있는 실정이다. 그러나 국내의 식물특이 당쇄구조 제어 기작 및 발현 연구는 아직 초기 단계에 있다. 모델 식물(애기장대)의 기초 유전자 연구와 당쇄 구조분석에 대한 기술은 확보되어 있으나 아직 외국의 선두 연구진과의 격차는 크다고 할 수 있다. 전세계적으로 많은 연구들을 통해 식물특이 당쇄구조인 α1,3-fucose, β1,2-xylose 및 Lewis-a epitope의 생합성을 제어할 수 있는 기술들이 확립되었고, α1,6-fucose나 β1,4-galactose와 같은 식물 N-glycan 내에 존재하지 않는 당쇄구조를 첨가하는 것도 가능해졌다. 최근 전세계적으로 활발하게 수행되고 있는 식물체 내에 sialic acid를 생합성하기 위한 대사 경로도 관련 효소들 및 transporter들의 연구 및 도입에 의해 구축되어가고 있다. 따라서 멀지 않은 미래에 인체와 동일한 N-glycan 구조를 가지는 당단백질을 대량 생산하는 식물체의 개발이 이루어질 것으로 전망되고, 유용 유전자의 형질전환을 통해 의료용 및 산업용 소재 개발에 활용됨으로써 장기적으로 동물세포배양 시스템을 대체할 수 있는 기술로 발전할 수 있을 것으로 기대된다. 이러한 인간화 당단백질을 생산하는 모델식물 시스템을 이용하여 고부가가치의 의약품 및 산업용 소재를 생산하는 분자농업 분야는 프로모터 연구를 통한 고발현 기술, 대사공학 관련 기술, 목적하는 세포 소기관으로의 집적 및 정제기술 등과 융합하여 미래의 바이오산업의 발전을 위한 핵심기술로 발전할 것으로 전망된다.

Mining of Serum Glycoproteins by an Indirect Approach Using Cell Line Secretome

Younghee Ahn,강운범,김준,이철주 한국분자세포생물학회 2010 Molecules and cells Vol.29 No.2

Glycosylation is the most important and abundant post-translational modification in serum proteome. Several specific types of glycan epitopes have been shown to be associated with various types of disease. Direct analysis of serum glycoproteins is challenging due to its wide dy-namic range. Alternatively, glycoproteins can be disco-vered in the secretome of model cell lines and then con-firmed in blood. However, there has been little experi-men-tal evidence showing cell line secretome as a tractable target for the study of serum glycoproteins. We used a hydrazine-based glycocapture method to selectively enrich glycoproteins from the secretome of the breast cancer cell line Hs578T. A total of 132 glycoproteins were identified by nanoLC-MS/MS analysis. Among the identified proteins, we selected 13 proteins that had one or more N-gly-cosylation motifs in the matched peptides, which were included in the Secreted Protein Database but not yet in the Plasma Proteome Database (PPD), and whose antibod-ies were commercially available. Nine out of the 13 se-lected proteins were detected from human blood plasma by western analysis. Furthermore, eight proteins were also detected from the plasma by targeted LC-MS/MS, which had never been previously identified by data-dependent LC-MS/MS. Our results provide novel proteins that should be enrolled in PPD and suggest that analysis of cell line secretome with subfractionation is an efficient strategy for discovering disease-relevant serum proteins.