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Yingxue Shao,이정환,추영국,고기성 한국곤충학회 2012 Entomological Research Vol.42 No.6
Colorectal cancer is the third most commonly diagnosed cancer in the world. Monoclonal antibody (mAb) CO17‐1A recognizes the tumor‐associated antigen GA733‐2, a cell surface glycoprotein highly expressed in colorectal carcinoma cells which is applicable for preventing and curing colorectal cancer. In this study, we tried to produce a new recombinant anti‐colorectal cancer large single chain (lsc) mAb based on mAb CO17‐1A in the baculovirus‐insect cell protein expression system. Two kinds of recombinant lsc mAbs were generated where variable light chain (VL) and heavy chain (HC) of mAb CO17‐1A were fused together by an interchain linker. The only difference between the two mAbs is based on fusion of an ER retention signal (KDEL) at its C‐terminus of HC. Polymerase chain reaction analysis verified the presence of both recombinant genes in the bacmid for generating viral expression vectors in insect cells. Western blot confirmed the expression of lsc mAbs in baculovirus‐infected insect cells. Cell enzyme linked immunosorbent assay (ELISA) showed that the mAbs from cell lysates bound to SW480 and SW620 human colorectal cancer cells. These results indicate that the baculovirus insect expression system can produce anti‐colorectal lsc mAb recognizing human colorectal cancer cells.
Yingxue Shao,Jeong-Hwan Lee,Deuk-Su Kim,Jun-Sik Ahn,Se-Ra Park,Youngtae Byun,Young-Kug Choo,Kisung Ko 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1
Colorectal cancer is the third most commonly diagnosed cancer in the world. Monoclonal antibody (mAb) CO17-1A recognizes the tumor-associated antigen GA733-2, a cell surface glycoprotein highly expressed in colorectal carcinoma cells which is applicable for preventing and curing colorectal cancer. In this study, we tried to produce a new recombinant anti-colorectal cancer large single chain (lsc) mAb based on mAb CO17-1A in the baculovirus-insect cell protein expression system. Two kinds of recombinant lsc mAbs were generated where variable light chain (VL) and heavy chain (HC) of mAb CO17-1A were fused together by an interchain linker. The only difference between two mAbs is depending of fusion of an ER retention signal (KDEL) at its C-terminus of HC. PCR analysis verified the presence of both recombinant genes in the bacmid for generating viral expression vectors insect cells. Western blot confirmed the expression of lsc mAbs in baculovirus-infected insect cells. Cell enzyme linked immunosorbent assay (ELISA) showed that the mAbs from cell lysates bound to SW480 and SW620 human colorectal cancer cells. These results indicate that the baculovirus insect expression system can produce anti-colorectal lsc mAb recognizing human colorectal cancer cells.
Zhe Lu,Yingxue Shao,Jung-Hwan Lee,Deuk-Soo Kim,Jun-Sik Yoon,Se-Ra Park,Kyung-Jin Lee,Doo-Byoung Oh,Kyung-A Hwang,Young-Kug Choo,Yeon Soo Han,Kisung Ko 한국당과학회 2011 한국당과학회 학술대회 Vol.2011 No.1
Colorectal cancer is the third leading cause of cancer-related death in the Korea. It is eager to find a method for preventing and curing colorectal cancer. The tumor-associated antigen GA733 is a cell surface glycoprotein highly expressed in colorectal carcinoma. In this study, GA733 was fused to Fc fragment of human IgG as a vaccine candidate in plant expression system. The fusion protein GA733-Fc can improve their expression in plant cells and induce cellular and humoral immune responses. GA733-Fc is glycosylated, however the N-glycan structures of plant-derived GA733-Fc differ from their parental counterparts. Plant-derived specific N-glycoforms have the potential to induce immunogenicity and allergenic responses in human therapy. Thus, oligomannose glycan structure was tried to be generated through retaining GA733-Fc and GA733 in ER with KDEL sequence. Agrobacterium-mediated transformation was conducted to generate transgenic plant expressing GA733-Fc and GA733. Recombinant protein expression, accumulation and biological activity of GA733-Fc with KDEL (GA733-FcK), GA733-Fc without KDEL (GA733-Fc), and GA733 with KDEL (GA733K) in transgenic plants were compared by western blot, confocal immunofluorescence analysis, ELISA and mouse immunization, respectively.
Expression of Anti-breast Cancer Monoclonal Antibody in Transgenic Plant
Kim, Deuk-Su,Shao, Yingxue,Lee, Jeong-Hwan,Yoon, Joon-Sik,Park, Se-Ra,Choo, Young-Kug,Hwang, Kyung-A,Ko, Ki-Sung The Korean Society of Environmental Agriculture 2011 한국환경농학회지 Vol.30 No.4
BACKGROUND: Plant expression system for mass production of recombinant proteins has several advantages over other existing expression systems with economical and safety issues. Breast cancer is a cancer originating from breast tissue and comprises almost 25% of all cancers in women world widely. Lewis-Y antigen is difucosylated oligosaccharide and is carried by glycoconjugates at cancer cell surface. In this study, the anti-breast cancer mAb BR55, which recognizes the epitope Lewis-Y, was expressed in the plant expression system. METHODS AND RESULTS: We have developed plant system for production of mAb BR55 with or without KDEL (the ER retention signal). This ER retention signal was attached to C-terminus of protein to help retain the recombinant glycoprotein carrying oligomannose glycans and enhance glycoprotein accumulation. PCR analysis was performed and confirmed the presence of recombinant genes. Western blot validated that the recombinant proteins mAb BR55 with or without KDEL were expressed in transgenic plants, moreover, the expression level of the mAb BR55 with KDEL was higher compared to the mAb BR55 without KDEL. CONCLUSION: These results indicate that KDEL fusion is a good way to produce proteins and plant can be an ideal expression system to obtain proteins and enhance accumulation of proteins.
Jeong-Hwan Lee,Yingxue Shao,Young-Kug Choo,Kyung-A Hwang,Kisung KO 한국당과학회 2013 한국당과학회 학술대회 Vol.2013 No.1
Colorectal cancer is the third most commonly diagnosed cancer in the world. Monoclonal antibody (mAb) CO17-1A recognizes the tumor-associated antigen GA733-2, a cell surface glycoprotein highly expressed in colorectal carcinoma cells which is applicable for preventing and curing colorectal cancer. In this study, we tried to produce a new recombinant anti-colorectal cancer large single chain (lsc) mAb based on mAb CO17-1A in the baculovirus-insect cell protein expression system. Two kinds of recombinant lsc mAbs were generated where variable light chain (VL) and heavy chain (HC) of mAb CO17-1A were fused together by an interchain linker. The only difference between the two mAbs is based on fusion of an ER retention signal (KDEL) at its C-terminus of HC. Polymerase chain reaction analysis verified the presence of both recombinant genes in the bacmid for generating viral expression vectors in insect cells. Western blot confirmed the expression of lsc mAbs in baculovirus-infected insect cells. Cell enzyme linked immunosorbent assay (ELISA) showed that the mAbs from cell lysates bound to SW480 and SW620 human colorectal cancer cells. These results indicate that the baculovirus insect expression system can produce anti-colorectal lsc mAb recognizing human colorectal cancer cells.
Jung-Hwan Lee,Deuk-Soo Kim,Yingxue Shao,Jun-Sik Yoon,Se-Ra Park,Yong-Seok Le,Yeon-Soo Han,Kyung-A Hwang,Young-Kug Choo,Kisung Ko 한국당과학회 2011 한국당과학회 학술대회 Vol.2011 No.1
In this study, we will attempt to analyze the ER localization, glycosylation pattern and anti-tumor activity of KDEL-fused monoclonal antibody. Advantages of the baculovirus insect cell expression system for production of recombinant proteins include high capacity, flexibility, and glycosylation capability. In this study, this expression system was exploited to produce anti-cancer monoclonal antibody (mAb) CO17-1A, which recognizes the antigen GA733 that is highly expressed on the surface membrane of colorectal carcinoma cells. The heavy chain (HC) and light chain (LC) genes of KDEL-fused mAb CO17-1A were cloned under the control of P10 and PPH promoters in the pFastBacTM dual vector, respectively. Gene expression cassettes carrying the HC and LC genes were transposed into a bacmid in Escherichia coli (DH10Bac). The transposed bacmid was transfected to Sf9 insect cells to generate baculovirus expressing mAb CO17-1A. In future, we will study the expression and localization of KDEL-fused mAb CO17-A in baculovirus infected cells by PCR, western blot, enzyme linked immunosorbent assay (ELISA), confocal immunofluorescence, fluorescence-activated cell sorting analysis (FACs) and antibody dependent cell cytotoxicity (ADCC).
Lu, Zhe,Lee, Kyung-Jin,Shao, Yingxue,Lee, Jeong-Hwan,So, Yangkang,Choo, Young-Kug,Oh, Doo-Byoung,Hwang, Kyung-A,Oh, Seung Han,Han, Yeon Soo,Ko, Kisung Hindawi Publishing Corporation 2012 Journal of biomedicine & biotechnology Vol.2012 No.-
<P>The tumor-associated antigen GA733 is a cell-surface glycoprotein highly expressed in colorectal carcinomas. In this study, 3 recombinant genes were constructed as follows: GA733 tagged to the ER retention sequence KDEL (GA733K), GA733 fused to the immunoglobulin Fc fragment (GA733-Fc), and GA733-Fc fused to the ER retention sequence (GA733-FcK). <I>Agrobacterium</I>-mediated transformation was used to generate transgenic plants expressing recombinant genes. The presence of transgenes was confirmed by genomic PCR. Western blot, confocal immunofluorescence, and sandwich ELISA showed the expression of recombinant proteins. The stability, flexibility, and bioactivity of recombinant proteins were analyzed and demonstrated through N-glycosylation analysis, animal trials, and sera ELISA. Our results suggest that the KDEL retained proteins in ER with oligomannose glycan structure and enhanced protein accumulation level. The sera of mice immunized with GA733-FcK purified from plants contained immunoglobulins which were at least as efficient as the mammalian-derived GA733-Fc at recognizing human colorectal cancer cell lines. Thus, a plant system can be used to express the KDEL fusion protein with oligomannose glycosylation, and this protein induces an immune response which is comparable to non-KDEL-tagged, mammalian-derived proteins.</P>