Expression of novel carbohydrate-specific single chain antibodies in E. coli and analyses of carbohydrate-binding activities of purified antibodies

• Project/Area Number: 22570125
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: Tokai University
• Principal Investigator: YAMAGUCHI Yoko 東海大学, 工学部, 教授 (10328106)
• Co-Investigator(Renkei-kenkyūsha): YAMAGUCHI Yoshiki 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)
• Project Period (FY): 2010 – 2012
• Project Status: Completed (Fiscal Year 2012)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2012: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2011: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2010: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: 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

Research Abstract

Aberration of carbohydrates on cell surface is often associated with pathogenic changes in diseases. Towards developing anti-cancer therapeutics, we had obtained genes for anti-carbohydrate single chain antibodies (scFvs) against carbohydrate moieties expressed specifically in cancer cells using phage-display methods. In this study, expression of scFv genes in E. coli and insect cells was successfully carried out to produce antibody proteins, whose carbohydrate binding activities were confirmed.

Research Products

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