新規糖鎖特異的単鎖抗体の大腸菌発現系による大量発現と糖鎖結合性解析

• 研究課題/領域番号: 22570125
• 研究種目: 基盤研究(C)
• 配分区分: 補助金
• 応募区分: 一般
• 研究分野: 構造生物化学
• 研究機関: 東海大学
• 研究代表者: 山口 陽子 東海大学, 工学部, 教授 (10328106)
• 連携研究者: 山口 芳樹 独立行政法人理化学研究所, システム糖鎖, チームリーダー (90323451)
• 研究期間 (年度): 2010 – 2012
• 研究課題ステータス: 完了 (2012年度)
• 配分額: 4,680千円 (直接経費: 3,600千円、間接経費: 1,080千円); 2012年度: 910千円 (直接経費: 700千円、間接経費: 210千円); 2011年度: 1,690千円 (直接経費: 1,300千円、間接経費: 390千円); 2010年度: 2,080千円 (直接経費: 1,600千円、間接経費: 480千円)
• キーワード: 大腸菌発現系 / 単鎖抗体 / 封入体 / リフォールディング / 糖鎖結合性 / 糖鎖親和性 / 昆虫細胞発現系

研究概要

タンパク質の50%以上が糖鎖で修飾されており、この糖鎖の変動が病態を反映することから、がんで露出する糖鎖に対する抗体作製を目指してきた。糖鎖に対する抗体は通常の免疫では取得困難なため、免疫法を体外で実現するファージ活用法でヒト型単鎖抗体を作製してきた。本研究では、取得した単鎖抗体の遺伝子からタンパク質をいかにして生産し、性質を見極めるかに重点を置き、単鎖抗体の大腸菌での大量生産・糖鎖結合活性の測定の確立と、昆虫細胞での生産にも成功した。

研究成果

• [雑誌論文] Effects of two monoclonal antibodies, MLS128 against Tn-antigen and 1H7 against insulin-like growth factor-I receptor, on the growth of colon cancer cells (2012)
  • 著者名/発表者名: Zamri, N., Masuda, N., Oura, F., Yajima, Y., Nakada, H., and Fujita-Yamaguchi, Y
  • 雑誌名: Biosci Trends 巻: 6 ページ: 303-312
  • 査読あり
• [雑誌論文] Overproduction of anti-Tn antibody MLS128 single-chain Fv fragment in Escherichia coli cytoplasm using a novel pCold-PDI vector (2012)
  • 著者名/発表者名: Subedi, G.P., Satoh, T., Hanashima,S., Ikeda, A., Nakada, H., Sato, R., Mizuno M., Yuasa, N., Fujita-Yamaguchi, Y., and Yamaguchi, Y.
  • 雑誌名: Protein Expr. Puri 巻: 82 ページ: 197-204
  • 査読あり
• [雑誌論文] Construction and expression of anti-Tn-antigen-specific single chain antibody genes from hybridoma producing MLS128 monoclonal antibody (2012)
  • 著者名/発表者名: Yuasa, N., Ogawa, H., Koizumi, T., Tsukamoto, K., Matsumoto-Takasaki, A., Asanuma, H., Nakada, H., and Fujita- Yamaguchi, Y.
  • 雑誌名: J Biochem 巻: 151 ページ: 371-381
  • 査読あり
• [雑誌論文] Surface plasmon resonance and NMR analyses of anti Tn-antigen MLS128 monoclonal antibody binding to two or three consecutive Tn-antigen cluster (2012)
  • 著者名/発表者名: Matsumoto-Takasaki, A., Hanashima, S.. Aoki, A., Yuasa, N., Ogawa, H., Sato, R., Kawakami, H., Mizuno, M. Nakada, H.,Yamaguchi, Y., and Fujita-Yamaguchi, Y
  • 雑誌名: J Biochem. 巻: 151 ページ: 273-282
  • NAID: 10031166066
  • 査読あり
• [雑誌論文] Effects of two monoclonal antibodies, MLS128 against Tn-antigen and 1H7 against insulin-like growth factor-I receptor, on the growth of colon cancer cells. (2012)
  • 著者名/発表者名: Zamri, N.
  • 雑誌名: BioScience Trends 巻: 6 ページ: 303-312
  • 査読あり
• [雑誌論文] Surface plasmon resonance and NMR analyses of anti Tn-antigen MLS128 monoclonal antibody binding to two or three consecutive Tn-antigen cluster (2012)
  • 著者名/発表者名: Matsumoto-Takasaki, A., Hanashima, S., Aoki, A., Yuasa, N., Ogawa, H., Sato, R., Kawakami, H., Mizuno, M., Nakada, H., Yamaguchi, Y., Fujita-Yamaguchi, Y.
  • 雑誌名: J Biochem.151 巻: 151 ページ: 273-282
  • NAID: 10031166066
  • 査読あり
• [雑誌論文] Construction and expression of anti-Tn-antigen-specific single chain antibody genes from hybridoma producing MLS128 monoclonal antibody (2012)
  • 著者名/発表者名: Yuasa, N., Ogawa, H., Koizumi, T., Tsukamoto, K., Matsumoto-Takasaki, A., Asanuma, H., Nakada, H., Fujita-Yamaguchi, Y.
  • 雑誌名: J Biochem.151. Feb 8 巻: 151(Epub ahead of print)(Not known yet)
  • 査読あり
• [雑誌論文] Overproduction of anti-Tn antibody MLS128 single-chain Fv fragment in Escherichia coli cytoplasm using a novel pCold-PDI vector (2012)
  • 著者名/発表者名: Subedi, G.P., Satoh, T., Hanashima, S., Ikeda, A., Nakada, H., Sato, R., Mizuno M., Yuasa, N., Fujita-Yamaguchi Y., Yamaguchi, Y.
  • 雑誌名: Protein Expr.Puri. 巻: 82 ページ: 197-204
  • 査読あり
• [雑誌論文] Characterization of three different single chain antibodies recognizing nonreducing terminal mannose residues expressed in Escherichia coli by an inducible T7 expression system (2011)
  • 著者名/発表者名: Matsumoto-Takasaki, A., Yuasa, N., Katagiri, D., Koyama, T., Sakai, K., Zamri, N., Phung, S., Chen, S., Nakada, H., Nakata, M., Fujita-Yamaguchi, Y.
  • 雑誌名: J Biochem 巻: 150 ページ: 439-450
  • 査読あり
• [雑誌論文] がん診断薬・抗体療法向け創薬を目指した糖鎖特異的ヒト型抗体の開発 (2011)
  • 著者名/発表者名: 山口(藤田)陽子、高崎(松本)綾乃、酒井恵子、湯浅徳行
  • 雑誌名: Journal of the Society of Japanese Women Scientists 巻: 11 ページ: 19-28
• [雑誌論文] Characterization of three different single chain antibodies recognizing non reducing terminal mannose residues expressed in Escherichia coli by an inducible T7 expression system (2011)
  • 著者名/発表者名: Matsumoto-Takasaki, A., Yuasa, N., Katagiri, D., Koyama, T., Sakai, K., Zamri, N., Phung, S., Chen, S., Nakada, H., Nakata, M., Fujita-Yamaguchi, Y.
  • 雑誌名: J Biochem.150 巻: 150 ページ: 439-450
  • 査読あり
• [雑誌論文] Production of anti-carbohydrate antibodies by phage-display technologies: Potential impairment of cell growth as a result of endogenous expression (2010)
  • 著者名/発表者名: Yuasa, N., Zhang, W., Goto, T., Sakaue, H., Matsumoto- Takasaki, A., Kimura, M., Ohshima, H., Tsuchida, Y., Koizumi, T., Sakai, K., Kojima, T., Yamamoto, K., Nakata, M., and Fujita-Yamaguchi, Y.
  • 雑誌名: J. Biol. Chem 巻: 285 ページ: 30587-30597
  • 査読あり
• [雑誌論文] Preparation of asialo-agalacto- glycophorin A for screening of anti-Tn antibodies (2010)
  • 著者名/発表者名: Koizumi, T., Matsumoto-Takasaki, A., Nakada, H., Nakata, M., Fujita-Yamaguchi, Y.
  • 雑誌名: BioScience Trends 巻: 4 ページ: 308-311
  • 査読あり
• [雑誌論文] Preparation of asialo-agalacto-glycophorin A for screening of and-Tn antibodies. (2010)
  • 著者名/発表者名: Koizumi, T., Matsumoto-Takasaki, A., Nakada, H., Nakata, M., Fujita-Yamaguchi, Y.
  • 雑誌名: Bioscience Trends 巻: 4 ページ: 308-311
  • 査読あり
• [雑誌論文] Production of anti-carbohydrate antibodies by phage-display technologies : Potential impairment of cell growth as a result of endogenous expression. (2010)
  • 著者名/発表者名: Yuasa, N., Zhang, W., Goto, T., Sakaue, H., Matsumoto- Takasaki, A., Kimura, M., Ohshima, H., Tsuchida, Y., Koizumi, T., Sakai, K., Kojima, T., Yamamoto, K., Nakata, M., Fujita-Yamaguchi, Y.
  • 雑誌名: J.Biol.Chem. 巻: 285 ページ: 30587-30597
  • 査読あり
• [学会発表] Effects of domain shuffling in VH-CDR3 between MLS128 and 83D4 on their Tn-antigen specificities (2012)
  • 著者名/発表者名: Nakazawa, A. et al
  • 学会等名: The 85th Annual Meeting of the Japanese Biochemical Society
  • 発表場所: Fukuoka
• [学会発表] Effects of anti-insulin-like growth factor-I receptor (IGF-IR) antibodies with or without KDEL ER-retention signal on cancer cell growth. (2012)
  • 著者名/発表者名: Guo, Y.-M., et al
  • 学会等名: The 85th Annual Meeting of the Japanese Biochemical Society
  • 発表場所: Fukuoka
• [学会発表] Purification and characterization of anti-T-antigen single chain antibody expressed the Drosophila Schneider 2 Cell System (2012)
  • 著者名/発表者名: Koyama, T. et al
  • 学会等名: The 85th Annual Meeting of the Japanese Biochemical Society
  • 発表場所: Fukuoka
• [学会発表] Refolding of E.coli expressed scFv antibody using lauroyl-L-glutamate (2012)
  • 著者名/発表者名: Fukuda, T. et al
  • 学会等名: The 85th Annual Meeting of the Japanese Biochemical Society
  • 発表場所: Fukuoka
• [学会発表] Effects of anti-insulin-like growth factor-I receptor (IGF-IR) antibodies with or without the ER-retention signal KDEL on cancer cell growth. (2012)
  • 著者名/発表者名: Guo, Y.-M. et al
  • 学会等名: 244th American Chemical Society National Meeting
  • 発表場所: Philadelphia, PA, USA
• [学会発表] In vitro production of anti-carbohydrate antibodies: Purification of anti-T antigen antibodies (scFvs) from E. coli inclusion bodies and Drosophila S2 cell culture media (2012)
  • 著者名/発表者名: Koyama, T. et al
  • 学会等名: 244th American Chemical Society National Meeting
  • 発表場所: Philadelphia, PA, USA
• [学会発表] Purification of anti-insulin-like growth factor-I receptor (IGF-IR) single chain antibody (scFv) expressed in Drosophila S2 cells (2012)
  • 著者名/発表者名: Nakazawa, A. et al
  • 学会等名: 244th American Chemical Society National Meeting
  • 発表場所: Philadelphia, PA, USA
• [学会発表] Expression, purification, and refolding of single chain antibodies (scFvs) from inclusion bodies. (2012)
  • 著者名/発表者名: Kato, H. et al
  • 学会等名: 244th American Chemical Society National Meeting
  • 発表場所: Philadelphia, PA, USA
• [学会発表] Expression, purification, and refolding of single chain antibodies (scFvs) from inclusion bodies. (2012)
  • 著者名/発表者名: Kato, N.
  • 学会等名: 244th American Chemical Society National Meeting
  • 発表場所: Philadelphia Convention Center (USA)
• [学会発表] Purification of anti-insulin-like growth factor-I receptor (IGF-IR) single chain antibody (scFv) expressed in Drosophila S2 cells. (2012)
  • 著者名/発表者名: Nakazawa, A.
  • 学会等名: 244th American Chemical Society National Meeting
  • 発表場所: Philadelphia Convention Center (USA)
• [学会発表] In vitro production of anti-carbohydrate antibodies: Purification of anti-T antigen antibodies (scFvs) from E. coli inclusion bodies and Drosophila S2 cell culture media. (2012)
  • 著者名/発表者名: .Koyama, T.
  • 学会等名: 244th American Chemical Society National Meeting
  • 発表場所: Philadelphia Convention Center (USA)
• [学会発表] Effects of anti-insulin-like growth factor-I receptor (IGF-IR) antibodies with or without the ER-retention signal KDEL on cancer cell growth. (2012)
  • 著者名/発表者名: Guo, Y.-M.
  • 学会等名: 244th American Chemical Society National Meeting
  • 発表場所: Philadelphia Convention Center (USA)
• [学会発表] Refolding of E.coli expressed scFv antibody using lauroyl-L-glutamate. (2012)
  • 著者名/発表者名: Fukuda, T.
  • 学会等名: The 85th Annual Meeting of the Japanese Biochemical Society
  • 発表場所: 福岡マリンメッセ
• [学会発表] Purification and characterization of anti-T-antigen single chain antibody expressed the Drosophila Schneider 2 Cell System. (2012)
  • 著者名/発表者名: Koyama, T.
  • 学会等名: The 85th Annual Meeting of the Japanese Biochemical Society
  • 発表場所: 福岡マリンメッセ
• [学会発表] Effects of anti-insulin-like growth factor-I receptor (IGF-IR) antibodies with or without KDEL ER-retention signal on cancer cell growth. (2012)
  • 著者名/発表者名: Guo, Y.-M.,
  • 学会等名: The 85th Annual Meeting of the Japanese Biochemical Society
  • 発表場所: 福岡マリンメッセ
• [学会発表] Effects of domain shuffling in VH-CDR3 between MLS128 and 83D4 on their Tn-antigen specificities. (2012)
  • 著者名/発表者名: Nakazawa, A.
  • 学会等名: The 85th Annual Meeting of the Japanese Biochemical Society
  • 発表場所: 福岡マリンメッセ
• [学会発表] NMR analysis of an anti-carbohydrate antibody MLS128 single-chain Fv fragment toward elucidation of the multivalent recognition mechanism. (2011)
  • 著者名/発表者名: Subedia, G.P. et al
  • 学会等名: The 50th Memorial Annual Meeting of the Nuclear Magnetic Resonance Society of Japan
  • 発表場所: Yokohama (Osanbashi Hall)
• [学会発表] NMR analysis of an anti-carbohydrate antibody MLS128 single-chain Fv fragment toward elucidation of the multivalent recognition mechanism (2011)
  • 著者名/発表者名: Subedia, G.P. et al
  • 学会等名: 2011 Annual Conference of the Society for Glycobiology
  • 発表場所: Seattle, WA, USA
• [学会発表] Purification and characterization of anti-Tn-antigen 4E10 single chain antibody expressed in E. coli (2011)
  • 著者名/発表者名: Fukuda, T. et al.
  • 学会等名: The 84th Annual Meeting of the Japanese Biochemical Society
  • 発表場所: Kyoto(京都国際会館)
• [学会発表] Purification and characterization of anti-T-antigen single chain antibody expressed in E. coli or the Drosophila Schneider 2 Cell System (2011)
  • 著者名/発表者名: Koyama, T. et al
  • 学会等名: The 84th Annual Meeting of the Japanese Biochemical Society
  • 発表場所: Kyoto(京都国際会館)
• [学会発表] Expression and purification of single chain antibodies (scFvs) isolated from a phage-displayed library with LNFPIII (Lex) probe (2011)
  • 著者名/発表者名: Kato, H. et al
  • 学会等名: American Chemical Society National Meeting and Exposition
  • 発表場所: Anaheim, CA, USA
• [学会発表] Purification and characterization of anti-LNFPIII single chain antibody (scFv) conjugated with Fc from CHO cells transfected with 3F1 scFv-Fc gene. (2011)
  • 著者名/発表者名: Tsuchida, Y. et al
  • 学会等名: 241st American Chemical Society National Meeting and Exposition
  • 発表場所: Anaheim, CA, USA
• [学会発表] In vitro production of anti-carbohydrate antibodies:Purification of anti-trimannose single chain antibodies (scFvs) from inclusion bodies (2011)
  • 著者名/発表者名: Katagiri, D. et al
  • 学会等名: 241st American Chemical Society National Meeting and Exposition
  • 発表場所: Anaheim, CA, USA
• [学会発表] Purification and characterization of anti-T-antigen single chain antibody expressed in E.coli or the Drosophila Schneider 2 Cell System (2011)
  • 著者名/発表者名: Koyama, T., Matsumoto-Takasaki, A., Yuasa, N., Katagiri, D., Fujita-Yamaguchi, Y.
  • 学会等名: The 84th Annual Meeting of the Japanese Biochemical Society
  • 発表場所: Kyoto(京都国際会館)
• [学会発表] Purification and characterization of anti-Tn-antigen 4E10 single chain antibody expressed in E.coli (2011)
  • 著者名/発表者名: Fukuda, T., Kawamoto, K., Mastumoto-Takasaki, A., Yuasa, N., Fujita-Yamaguchi, Y.
  • 学会等名: The 84th Annual Meeting of the Japanese Biochemical Society
  • 発表場所: Kyoto(京都国際会館)
• [学会発表] NMR analysis of an anti-carbohydrate antibody MLS128 single-chain Fv fragment toward elucidation of the multivalent recognition mechanism (2011)
  • 著者名/発表者名: Subedia, G.P., Satoh, T., Hanashima, S., Ikeda, A., Nakada, H., Sato, R., Mizuno, M., Yuasae, N., Fujita-Yamaguchi, Y., Yamaguchi, Y.
  • 学会等名: The 50th Memorial Annual Meeting of the Nuclear Magnetic Resonance Society of Japan
  • 発表場所: Yokohama (Osanbashi Hall)
• [学会発表] In vitro production of anti-carbohydrate antibodies : Purification of anti-trimannose single chain antibodies (scFvs) from inclusion bodies (2011)
  • 著者名/発表者名: Daiichi Katagiri, Tubasa Koyama, Ayano Matsumoto-Takasaki, Nobuyuki Yuasa, Munehiro Nakata, Yoko Fujita-Yamaguchi
  • 学会等名: 241^<st> American Chemical Society National Meetingand Exposition
  • 発表場所: Anaheim, CA, USA
• [学会発表] Purification and characterization of anti-LNFPIII single chain antibody (scFv) conjugated with Fc from CHO cells transfected with 3F1 scFv-Fcgene (2011)
  • 著者名/発表者名: Yasunobu Tsuchida, Kaname Funayama, Daisuke Kubo, Noriyuki Yuasa, Yoko Fujita-Yamaguchi
  • 学会等名: 241^<st> American Chemical Society National Meeting and Exposition
  • 発表場所: Anaheim, CA, USA
• [学会発表] Expression and purification of single chain antibodies (scFvs) isolated from aphage-displayed library with LNFPIII (Le^x) probe (2011)
  • 著者名/発表者名: Hiroyuki Kato, Haruhiko Ogawa, Masato Takano, Daiichi Katagiri, Kotaro Kawamoto, Noriyuki Yuasa, Ayano Matsumoto-Takasaki, Yoko Fujita-Yamaguchi
  • 学会等名: 241^<st> American Chemical Society National Meeting and Exposition
  • 発表場所: Anaheim, CA, USA
• [学会発表] Establishment of overproduction procedure for anti-Tn antigen MLS128 single-chain Fv fragment toward the structural studies (2010)
  • 著者名/発表者名: Subedi, G. P., Satoh, T., Hanashima, S., Ikeda, A, Nakada, H., Sato, R., Mizuno, M., Yuasa, N., Fujita-Yamaguchi, Y., and Yamaguchi, Y.
  • 学会等名: BMB2010 (日本生化学会・分子生物学会)
  • 発表場所: 神戸ポートアイランド(兵庫県)
• [学会発表] Purification and characterization of anti-LNFPIII antibody expressed in CHO cells (2010)
  • 著者名/発表者名: Tsutida, Y. et al
  • 学会等名: BMB2010 (日本生化学会・分子生物学会)
  • 発表場所: 神戸ポートアイランド(兵庫県)
• [学会発表] Refolding and characterization of three anti-mannotriose single chain antibodies (2010)
  • 著者名/発表者名: Katagiri, D. et al
  • 学会等名: BMB2010 (日本生化学会・分子生物学会)
  • 発表場所: BMB2010 (日本生化学会・分子生物学会)
• [学会発表] Construction and expression of anti-Tn-antigen (GalNAca-Ser/Thr)-specific MLS128 single chain antibody (scFv) genes (2010)
  • 著者名/発表者名: Ogawa, H. et al
  • 学会等名: 240th American Chemical Society National Meeting and Exposition
  • 発表場所: Boston, MA, USA
• [学会発表] In vitro production of anti-carbohydrate antibodies: Purification of anti- T-antigen (beta Gal1-3GalNAc-Thr/Ser) single chain antibodies (scFvs) from inclusion bodies (2010)
  • 著者名/発表者名: Matsumoto-Takasaki, A. et al
  • 学会等名: 240th American Chemical Society National Meeting and Exposition
  • 発表場所: Boston, MA, USA
• [学会発表] In vitro production of anti-carbohydrate antibodies : Purification of anti-T-antigen (beta Gall-3GalNAc-Thr/Ser) single chain antibodies (scFvs) from inclusion bodies. (2010)
  • 著者名/発表者名: Matsumoto-Takasaki, A., Yamaguchi, S., Koizumi, T., Yuasa, N., Fujita-Yamaguchi, Y.
  • 学会等名: 240^<th> American Chemical Society National Meeting and Exposition
  • 発表場所: Boston, MA, USA
• [学会発表] Refolding and characterization of three anti-mannotriose single chain antibodies (2010)
  • 著者名/発表者名: Katagiri, D., Matsumoto-Takasaki, A., Yuasa, N., Koyama, T., Ogawa, H. Sakai, K., Takayanagi, A., Nakata, M., Fujita-Yamaguchi, Y.
  • 学会等名: BMB2010(日本生化学会・分子生物学会)
  • 発表場所: 神戸ポートアイランド(兵庫県)
• [学会発表] Purification and characterization of anti-LNFPIII antibody expressed in CHO cells (2010)
  • 著者名/発表者名: Tsutida, Y., Yuasa, N., Goto, T., Kubo, T., Zhang, W., Fujita-Yamaguchi, Y.
  • 学会等名: BMB2010(日本生化学会・分子生物学会)
  • 発表場所: 神戸ポートアイランド(兵庫県)
• [学会発表] Establishment of overproduction procedure for anti-Tn antigen MLS128 single-chain Fv fragment toward the structural studies. (2010)
  • 著者名/発表者名: Subedi, G. P., Satoh, T., Hanashima, S., Ikeda, A, Nakada, H., Sato, R., Mizuno, M., Yuasa, N., Fujita-Yamaguchi, Y., Yamaguchi, Y.
  • 学会等名: BMB2010(日本生化学会・分子生物学会)
  • 発表場所: 神戸ポートアイランド(兵庫県)