• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to project page

1998 Fiscal Year Final Research Report Summary

CELL-FREE BIOSYNTHESIS SYSTEM OF NOVEL ANTIBODIES

Research Project

Project/Area Number 09044119
Research Category

Grant-in-Aid for international Scientific Research

Allocation TypeSingle-year Grants
SectionJoint Research
Research Field Molecular biology
Research InstitutionTOHOKU UNIVERSITY

Principal Investigator

KUMAGAI Izumi  TOHOKU UNIVERSITY,PROFESSOR, 大学院・工学研究科, 教授 (10161689)

Co-Investigator(Kenkyū-buntansha) VOLKER Erdma  ベルリン自由大学, 生物化学研究所, 教授
ERDMANN Volker A.  FREIE UNIVERSITAT BERLINE,PROFESSOR
Project Period (FY) 1997 – 1998
KeywordsANTIBODY / CELL-FREE BIOSYNTHESIS SYSTEM / MOLECULAR RECOGNITION / GENETIC ENGINEERING / ANTIGEN SPECIFICITY / PROTEIN ENGINEERING / EVOLUTIONARY ENGINEERING
Research Abstract

Single-chain antibodies (scFv), which can be produced in Escherichia coli cells, have been shown in numerous cases to be active in antigen binding. In the case of the two anti-lysozyme single-chain antibodies, scFvLH and scFvHL, which have the reversed arrangement of the variable domains of the heavy and light chains of the corresponding monoclonal antibodies, the expression level differs greatly when they are produced in Escherichia coli [Tsumoto et al. (1995) Biochem. Biophys. Res. Commun. 201, 546-551]. Although the expression level of scFvLH is high in vivo, the single chain antibody with the reversed orientation (scFvHL) was synthesized in a very low yield and no active product could be obtained. In this study, we constructed the synthesis system of these two anti-lysozyme single-chain antibodies in high yields and with high biological activities in a cell-free E.coli expression system in the presence of reduced and oxidized glutathione, protein disulfide isomerase (PDI), and chap … More erones. In immunological blotting assays, the synthesized scFvs with both arrangements exhibit specific binding activity to the corresponding antigens, hen egg-white lysozyme, and in an activity assay both inhibited the action of lysozyme. scFvLH is synthesized mainly as a product with the expected molecular weight, whereas scFvHL is produced with additional shorter fragments, suggesting that the low yield isolation through the expression in vivo is due to mistranslation or ribonucleolytic cleavage of the transcript, In the cell-free expression of scFv a certain amount of the product is precipitated but in the presence of chaperones the amount of soluble protein increased from 25 to 90% (PDI and chaperones). The overall expression level and the specific biological activity, however, were hardly influenced. The system reported here can provide significant amounts of various scFv fragments regardless of the order of variable regions, including those which are hardly expressed in vivo. Based on the cell-free synthesis system, the expression of ribosomal protein 57 fused with HyHEL10 scFv LH was attempted, and the technical basis for molecular evolution of proteins has been constructed. Less

  • Research Products

    (38 results)

All Other

All Publications (38 results)

  • [Publications] Tsumoto et al.: "Novel selection method for engineered antibodies using the mechanism of FV fragment stabilization in the presence of antigen" Protein Engineering. 10. 1311-1318 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Furuta et al.: "Construction of mono-and bi-valent human single-chain Fv fragments against the O-antigen in the Rh blood group" Protein Engineering. 11. 233-241 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tsumoto et al.: "Highly efficient recovery of functional single-chain for fragment from inclusion bodies overexpressed in E col: by controlled・・・・・" Journal of Immunological Methods. 219. 119-120 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Koshiba et al.: "Thermal study of mutant human lysozymes with partially introduced Cu^<2+> binding sites by using efficient refolding system" Protein Engineering. 11. 583-590 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Chaudhuri et al.: "Effect of the extra N-terminal methionire residue on the stability and folding of recombinant α-lactalbumis in E,coli" Journal of Molecular Biology. 285. 1179-1199 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Merk et al.: "Cell-free expression of two single-chain monoclonal antibodies against lysozyme" Journal of Biochemistry. 125. 328-331 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 前仲ら: "Structural analysis of mutant hen egg-white lysozyme prefering a minor binding mode" Biochin. Biophys. Acta. 1384. 23-31 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kitamura et al.: "Cloning and expression of the gene encoding flavodoxin from Desulfovibrio valgaris (Miyazaki F)" Journal of Biochemistry. 123. 891-898 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Maenaka et al.: "Disclosure of another carbohydrate binding site in hen egg-white lysozyme by conversion of subsite B stmcture" Biochemical Journal. 331. 71-76 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hori et al.: "Substrate Recognitim of tRNA(Guanosine-2′-)-methyltransferase from Thermus thermophilus HB 27" Journal of Biological Chemistry. 273. 25721-25727 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tanaka et al.: "Pl-Specificity of Aqualysin I (a Subtilisin-type Serine Protease) from Thermus aquaticus YT-1, using Pl-Substituted Deriratives of SSI" Biosci. Biotechnol. Biochem.62. 2035-2038 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sakurai et al.: "SEA-scFv as bifunctional antibody." Biochemical Biophysical Research Communication. 印刷中. (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Suzuki et al.: "Open sandwich ELISA with VH-NL-alkaline phosphatase fusion proteins" Journal of Immunological Methods. 印刷中. (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kitamura et al.: "Expression of a Model Peptide of a Marine Mussel Adhesive Protein in E.coli." Journal of Polymer Science, A, Polymer Chemistry. 印刷中. (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 熊谷,津本: "生体外選択系による新機能抗体分子の創製" 蛋白質核酸酵素. 43. 159-167 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 熊谷,津本,前仲: "ファージ提示系による抗体工学" 日本生物物理学会誌. 213. 1219-1222 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 上田ら: "新たな免疫測定法" 遺伝子医学. 3. 170-175 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 熊谷, 津本, 前仲: "構造生物学(共著)" 朝倉出版, 13 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 熊谷, 津本: "遺伝子工学(分子遺伝学 共著)" 丸善出版社, 25 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 三沢, 熊谷: "医薬品開発を目指したプロテインエンジニアリングの現状(共著)" CMC 出版, 8 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 熊谷, 津本: "抗体工学(天然高分子研究の新展開,共著)" CMC 出版, 12 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Helmut Merk, Wolfgang Stiege, Kouhei Tsumoto, Izumi Kumagai, and Volker A.Erdmann: "Cell-free expression of two single-chain monoclonal antibodies against lysozyme-Effect of domain arrangement on the expression-" J.Biochem.125. 328-333 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kouhei Tsumoto, Yoshiyuki Nishimiya, Hiroshi Ueda, Teruyuki Nagamune, Kyoko Ogasahara, Katsuhide Yutani, Kenji Tokuhisa, Masaaki Matsushima and Izumi Kumagai: "Novel selection method for engineered antibodies using the mechanism of Fv fragment stabilization in the presence of antigen" Protein Engineering. 10. 1311-1318 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Masaru Furuta, Makoto Uchikawa, Yoshitaka Ueda, Toshio Yabe, Tetsuya Taima, Kouhei Tsumoto, Shusei Kojima, Takeo Juji and Izumi Kumagai: "Construction of mono- and bi- valent human single-chain Fv fragments against the D-antigen in the Rh blood group : Multimerization effect on cell agglutionation and application to blood typing" Protein Engineering. 11. 233-241 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kouhei Tsumoto, Katsutoshi Shinoki, Hidemasa Kondo, Makoto Uchikawa, Takeo Juji, and Izumi Kumagai: "High efficient recovery of functional single-chain Fv fragment from inclusion bodies overexpressed in E.coli by controlled introduction of oxidizing reagent---Its application to a human single-chain Fv---" J.Immunol.Methods. 219. 119-129 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Chikako Suzuki, Hiroshi Ueda, Kouhei Tsumoto, Walt C.Mahoney, Izumi Kumagai, and Teruyuki Nagamune: "Open Sandwich ELISA with VH-/VL-alkaline phosphatase fusion proteins" J.Immunol.Methods. (in press). (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Naoki Sakurai, Toshio Kudo, Masanori Suzuki, Kouhei Tsumoto, Shinichi Takemura, Hideaki Kodama, Shinji Ebara, Atsushi Teramae, Yu Katayose, Masao Shinoda, Tadashi Kuroki, Yuji Hinoda, Kohzoh Imai, Seiki Matsuno, and Izumi Kumagai: "SEA-scFv as bifunctional antibody : construction of the bacterial expression system and its functional analysis" Biochem.Biophys.Res.Commun.(in press). (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hiroshi Ueda, Masahiro Kawahara, Kouhei Tsumoto, Kazuo Tadokoro, Eiji Suzuki, Hajime Nishimura, Paula A.Schueler, Greg Winter, Walt Mahoney, Izumi Kumagai, and Teruyuki Nagamune: "Controlling cell growth by monovalent antigen" J.Mol.Biol.(in press). (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Masaya Kitamura, Yuichi Koshino, Yoshitaka Kamikawa, Kyoko Kohono, Shuichi Kojima, Kin-ichiro Miura, Takamasa Sagara, Hideo Akutsu, Izumi Kumagai, and Tadao Nakaya: "Cloning and expression of the Rubredoxin gene from Desulfovibrio vulgaris (Miyazaki F) -Comparison of the primary structure of desulfoferrodoxin-" Biochim.Biophys.Acta. 1351. 239-247 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Masaya Kitamura, Takamasa Sagara, Masahiro Taniguchi, Masaki Ashida, Kaori Ezoe, Kyoko Kohono, Shuichi Kojima, Kiyoshi Ozawa, Hideo Akutsu, Izumi Kumagai, and Tadao Nakaya: "Cloning and Expression of the Gene Encoding Flavodoxin from Desulfovibrio vulgaris (Miyazaki F)" J.Biochem.123. 891-898 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Katsumi Maenaka, Masaki Matsushima, Kimitsuna Watanabe, and Izumi Kumagai: "Structural analysis of mutant hen egg-white lysozyme prefering a minor binding mode." Biochim.Biophys.Acta. 1384. 23-31 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Katsumi Maenaka, Masaki Matsushima, Gota Kawai, Akinori Kidera, Kimitsuna Watanabe, Ryota Kuroki and Izumi Kumagai: "Disclosure of another carbohydrate binding site in hen egg-white lysozyme by conversion of subsite B structure" Biochem, J.331. 71-76 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Takumi Koshiba, Kouhei Tsumoto, Kazuo Masaki, Keiichi Kawano, Katsutoshi Nitta, and Izumi Kumagai: "Thermal study of mutant human lysozymes with partially introduced Ca ^<2+> binding sites by using efficient refolding system from inclusion bodies" Protein Engineering. 11. 583-590 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] H.Hori, S.Nakanishi, K.Nishikawa, T.Ueda, I.Kumagai, K.Watanabe: "Substrate Recognition of tRNA (Guanosine-2'-) -methyltransferase from Thermus thermophilus HB27" J.Biol.Chem.273. 25721-25727 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Terumichi Tanaka, Hiroshi Matsuzawa, Shuichi Kojima, Izumi Kumagai, Kin-ichiro Miura, and Takahisa Ohta: "P1-Specificity of Aqualysin I (a Subtilisin-type Serine Protease) from Thermus aquaticus YT-1, using P1-Substituted Derivatives of Streptomyces Subtilisin Inhibitor." Biosci.Biotechnol.Biochem.62. 2035-2038 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Chaudhuri, Tapan K., Katsunori Horii, Takao Yoda, Munehito Arai, Shunzi Nagata, Tokoki P.Terada, Hidefumi Uchiyama, Teikichi Ikura, Kouhei Tsumoto, Hiroshi Kataoka, Masaaki Matsushima, Kunihiro Kuwajima, Izumi Kumagai: "Effect of the extra N-terminal methionine residue on the stability and folding of recombinant alpha-lactabumin expressed in Escherichia coli" J.Mol.Biol.(in press). (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Masaya Kitamura, Kiminori Kawakami, Naotoshi Nakamura, Kouhei Tsumoto, Hidefumi Uchiyama, Yoshitaka Ueda, Izumi Kumagai, and Tadao Nakaya: "Expression of a Model Peptide of a Marine Mussel Adhesive Protein in Escherichia coli and Characterization of its Structural and Functional Properties" J.Polymer Sci, A,Polymer Chemstry. (in press). (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Izumi Kumagai and Kouhei Tsumoto: "Antigen-antibody binding" Encyclopedia of life sciences. (in press). (1999)

    • Description
      「研究成果報告書概要(欧文)」より

URL: 

Published: 1999-12-08  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi