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Effect of ER signal peptides (KDEL) on expression and function of monoclonal antibody in plant
Da-Young Park,Eun-Yi Oh,Zhe Lu,Yangkang So,Mi-Hyun Ahn,Mira Song,Kyung-Jin Lee,Doo-Byoung Oh,Youngkwan Kim,Kisung Ko 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1
Plant genetic engineering led to the production of plant-derived mAb (mAbP), which provides a safe and economically feasible alternative to the current animal expression systems. In this study, the expression level of mAbP SO57 between with and without Lys-Asp-Glu-Leu (KDEL) was compared in transgenic plant. PCR and PT-PCR analyse showed stable gene insertion transcription of heavy and light chain genes of mAbP SO57 with or without KDEL in plant, respectively. We validated expression of mAbP SO57 by western blot. Western blot showed the significantly higher expression level of mAbP SO57 with KDEL compared to without KDEL. Flow cytometry (FACS) analysis showed that the Fc domains of both purified mAbP and mammalian-derived mAb (mAbM) evidenced similar binding activity to the FcγRI receptor (CD64). High performance liquid chromatography (HPLC) analysed, glycosylation patterns of mAbP SO57 with or without KDEL. The mAbP SO57 with KDEL had glycan profile with both oligomannose type (47.6%) and golgi type (52.4%), while the mAbP SO57 without KDEL had only golgi type (100%) glycans. Neutralizing analysis with rabies virus CVS-11 showed the similar neutralizing activity between mAbP SO57 with and without KDEL. These results suggest the potential of mAbP SO57 for rabies immunotherapy, regardless of plant specific glycan structures.
Da-Young Park,Yangkang So,Kyung-Jin Lee,Zhe Lu,Eun-Yi Oh,Kwang-Wok Min,Seul-Ki Lee,Jung-Hwan Lee,Mi-Hyun Ahn,Mira Song,Doo-Byoung Oh,Youngkwan Kim,Kisung Ko 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1
Plant genetic engineering has led to the production of plant-derived mAb (mAbP), which provides a safe and economically feasible alternative to the current antibody expression systems. In this study, the expression levels of mAbP SO57 with or without ER-signal peptides (Lys-Asp-Gly-Leu;KDEL) in transgenic tobacco plants were analysed in transgenic plant. PCR and Reverse Transcription-PCR analyses showed existence of heavy and light chain genes of mAb with or without KEDL and their transcription in plant, respectively. Western blot showed that the expression levels of mAbP SO57 with KDEL were significantly higher than that without KDEL. Flow cytometry analysis showed that the Fc domains of both purified mAbP and mammalian-derived mAb have similar binding activity to the FcγRI receptor. High-performance liquid chromatography analysis showed that the mAbP SO57 with KDEL had glycan profile with both oligomannose and golgi type, whereas the mAbP SO57 without KDEL had only golgi type glycans. Neutralizing analysis with rabies virus CVS-11 showed the similar neutralizing activity between mAbP SO57 with and without KDEL. These results suggest that the potential of mAbP SO57 for rabies immunotherapy is regardless of plant specific glycan structures.
Da-Young Park,Kyung-Jin Lee,Yangkang So,Zhe Lu,Seulki Lee,Jung-Hwan Lee,Seung Han Oh,Yeon Soo Han,Doo-Byoung Oh,Youngkwan Kim,Kisung Ko 한국당과학회 2010 한국당과학회 학술대회 Vol.2010 No.1
Plant genetic engineering has led to production of plant-derived mAb (mAbP), which can provides a safe and economically effective alternative to the conventional antibody expression methods. In this study, the expression levels of mAbP SO57 with or without ER-retention peptide extensions signal (Lys-Asp-Glu-Leu; KDEL) in transgenic tobacco plants were analysed in transgenic plant. The expression levels of mAbP SO57 with KDEL were significantly higher than that without KDEL regardless of transcription level. mAbP SO57 with KDEL localized surround to the nucleus suggesting that the mAbP with KDEL is localized in ER. The mAbP without KDEL and mAbH had mainly Golgi type glycans, whereas the ER-localized mAbP with KDEL had glycan profile with both oligomannose type (47.6%) and Golgi type (52.4%). The Fc domains of both purified mAbP (with and without KDEL) and human-derived mAb (mAbH) had similar bingding activity to the Fcγ RI receptor (CD64). Both mAbP (with and without KDEL) had a shorter half-life than mAbH. However, both mAbP with and without KDEL was as effective at neutralizing activity of the rabies virus CVS-11 as the mAbH. These results suggest that ER localization of recombinant mAbP by KDEL reprograms glycosylation and enhances production of the functional antivirus therapeutic antibody in the plant.
Yangkang So,Seul-Ki Lee,Da-Young Park,Zhe Lu,Eun-Yi Oh,Kwang-Wook Min,Jung-Hwan Lee,Mi-Hyun Ahn,Mira Song,Kyung-Jin Lee,Doo-Byoung Oh,Youngkwan Kim,Kisung Ko 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1
The mAb CO17-1A recognizes the colorectal antigen GA733-2E, which highly is expressed on the surface membrane of human colorectal carcinoma cells. The higher yield of CO17-1A mAb reflect the use of the ER retrieval motif KDEL(Lys-Asp-Glu-Leu), In this study, the expression level of mAb CO17-1A was compared between mAbs fused with and without KDEL in plant. Western blot showed that the expression levels of mAb CO17-1A with KDEL were significantly higher than that without KDEL Cell ELISA with human colorectal carcinoma cell line confirmed that expression of the mAb with KDEL was significantly higher than that of mAb without KDEL. Flow cytometry analysis showed that the Fc domains of both purified mAbP (with and without KDEL) and mammalian-derived mAb have similar binding activity to the FcγRI receptor. The glycosylation analysis revealed that mAb CO17-1A without KDEL had plant specific glycans structure while mAb CO17-1A with KDEL had mainly oligomannose type glycans. Taken together, these studies suggest that KDEL affects expression of mAb and its glycan structures.
Expression of Anti-Colorectal Cancer Immunotherapeutic Monoclonal Antibody in Transgenic Plant
Yangkang So,Zhe Lu,Eun-Yi Oh,Da-Young Park,Kyung-Jin Lee,Mira Song,Seung Han Oh,Arshad Jamal,Mi-Hyun Ahn,Yeon Soo Han,Doo-Byoung Oh,Youngkwan Kim,Kisung Ko 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1
The mAb CO17-1A recognizes the colorectal antigen GA733-2E, which highly is expressed on the surface membrane of human colorectal carcinoma cells. We have successfully developed plant system for production of anti-colorectal cancer monoclonal antibody, mAb CO17-1A. Plant expression system for mAb production has several advantages in terms of the lack of animal pathogenic contaminants, low cost, ease of scale-up for agricultural production, and efficient post-translational glycosylation modifications compared to the conventional fermentation methods. KDEL (Lys-Asp-Glu-Leu), ER retention signal attached to C-terminus of protein might induce high protein expression and the glycoprotein carrying oligomannose glycans. In this study, the expression level of mAb CO17-1A was compared between mAbs fused with and without KDEL in plant. PCR analysis showed stable gene insertion of heavy and light chain genes of mAb CO17-1A with or without KDEL. We validated the expression of mAb CO17-1A by western blot and its subcellular localization under the confocal microscopy. Western blot showed the significantly increased expression level of mAb CO17-1A with KDEL compared to without KDEL. Cell ELISA with human colorectal carcinoma cell line confirmed that expression of the mAb with KDEL was significantly higher than that of mAb without KDEL. The glycosylation analysis revealed that mAb CO17-1A without KDEL had plant specific glycans structure while mAb CO17-1A with KDEL had mainly oligomannose type glycans. Taken together, these studies suggest that KDEL affects expression of mAb and its glycan structures.
레몬그라스와 자소엽 추출물의 피부보습 및 피부장벽에 관한 연구
소양강 ( Yangkang So ),황지영 ( Ji Young Hwang ),김현우 ( Hyun Woo Kim ),조하늘 ( Ha Neul Jo ),이태범 ( Tae-bum Lee ) 대한화장품학회 2019 대한화장품학회지 Vol.45 No.3
레몬그라스와 자소엽 추출물은 다양한 생리 효과를 나타내는 것으로 알려져 있지만, 피부보습과 피부장벽에 미치는 영향은 아직까지 연구되지 않았다. 본 연구에서는 레몬그라스와 자소엽 추출물의 피부보습과 피부장벽에 미치는 영향과 페놀성 화합물을 분석하였다. 피부각질형성세포에서 각 추출물이 피부보습에 미치는 영향을 조사한 결과, 두 추출물 모두 물보다 에탄올 추출물에서 히알루론산 생성이 많았다. HPLC를 이용한 19종 페놀성 화합물 분석 결과는 레몬그라스 에탄올 추출물(CCE)에서 chlorogenic acid와 p-coumaric acid가 검출되었으며 자소엽 에탄올 추출물(PFE)에서 rosmarinic acid와 caffeic acid가 검출되었다. 피부보습에 관련된 HAS1, HAS2, HAS3 및 AQP3와 피부장벽에 관련된 filaggrin, loricrin 발현은 PFE보다 CCE에서 높았다. 또한, CCE, PFE 모두 피부보습과 표피분화 조절에 관여하는 PPAR-α 단백질의 발현이 농도 의존적으로 증가하였으며 CCE의 주요성분인 chlorogenic acid와 p-coumaric acid가 PPAR-α 발현을 증가시켰다. 결론적으로 피부보습과 피부장벽보호 효과에 있어서 CCE가 PFE보다 우수한 효과를 나타내었고, 두 추출물은 피부보습과 피부장벽개선에 대한 기능성 소재로써 활용될 수 있을 것이라 생각된다. Cymbopogon citratus (CC) and Perilla frutescens (PF) are known to exert various biological effects. However, their skin hydration and skin barrier effects remain unclear. This study investigated effects of their extracts on skin hydration and skin barrier and analysed the phenolic compounds. effects of these extracts on skin hydration in HaCaT cells showed that Hyaluronic acid production in cells treated with ethanol extracts was higher than that treated with water extracts for both CC and PF. HPLC was used to analyse 19 phenolic compounds in CC and PF ethanol extracts (CCE and PFE). Results revealed chlorogenic acid and p-coumaric acid in CCE and rosmarinic acid and caffeic acid in PFE. Expression levels of hyaluronan synthase 1 (HAS1), HAS2, HAS3, and aquaporin 3 (AQP3), which are related to skin moisturization, and filaggrin and loricrin, which are related to skin barrier were higher in cells treated with CCE than with PFE. CCE and PFE also increased expression of PPAR-a protein involved in skin moisturization and epidermal differentiation in a concentration-dependent manner. As major components of CCE, chlorogenic acid and p-coumaric acid increased PPAR-a protein expression. Thus, CCE and PFE could be used as functional cosmetic materials for skin hydration and skin barrier effects.
Protein expression condition optimization of Baculovirus Expression System
Da-young Park,Yangkang So,Kisung Ko 한국당과학회 2010 한국당과학회 학술대회 Vol.2010 No.1
Baculovirus expression system is considered to have several benefits for production of recombinant protein such as high capacity, flexibility, and similar human glycosylation pattern. Typically, before any large scale expression work was commenced, an accurate titering of recombinant baculovirus is required to ensure the most appropriate expression conditions. One of them, the method have been developed by BaculoELISA Titer Kit (Clontech, Mountain Biew, CA, USA). Stocks of any Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) type baculovirus can be titered using this kit. However, control virus of this kit is high cost and relatively small quantity in kit. In this study, production of mAb CO17-1A by baculovirus infection was performed at different times, concentrations and passages. Immunoblot analysis confirmed expression of mAb CO17-1A in the infected cells. mAb CO17-1A expressed significantly high level under the condition of baculovirus passages 3, 72h and 400㎕ in 5ml insect cells after infection. The results indicated that it is not necessary to use BaculoELISA assay.
Induction of apoptosis by 2,3-dehydrosilybin via a caspase-dependent pathway in human HeLa cells.
Cho, Byoung Ok,So, Yangkang,Jin, Chang Hyun,Byun, Myung Woo,Seo, Kwon Il,Ko, Kisung,Chun, Myoung Sook,Jeong, Il Yun Japan Society for Bioscience, Biotechnology, and A 2014 Bioscience, Biotechnology, and Biochemistry Vol.78 No.2
<P>The aim of this study was to investigate the mechanisms involved in the apoptosis of HeLa cells due to 2,3-dehydrosilybin (DHS) treatment. DHS treatment over 24 h significantly inhibited cell viability and induced apoptosis in a dose-dependent manner. It also triggered the cleavage of caspase-8, caspase-9, caspase-3, and PARP, and significantly increased caspase-3 activity in a dose-dependent manner. Moreover, it triggered the depolarization of the mitochondrial membrane potential (δψm), the release of cytochrome c into the cytosol, the cleavage of Bid, and the downregulation of Bcl-2 in a dose-dependent manner. Furthermore, z-VAD-fmk (a pan-caspase inhibitor) and z-IETD-fmk (a specific caspase-8 inhibitor) abolished the DHS-induced activation of the caspase-8, -9, and -3, cleavage of PARP, the depolarization of δψm, the release of cytochrome c, the cleavage of Bid, and the downregulation of Bcl-2. Taken together, these results suggest that DHS-induced apoptosis is mediated by a caspase-dependent pathway in human HeLa cells.</P>