1998 Fiscal Year Annual Research Report

新機能抗体-受容体キメラ蛋白質の創製と細胞増殖制御機構解析への応用

Research Project

Project/Area Number	10179202
Research Institution	Tohoku University
Principal Investigator	熊谷泉東北大学, 大学院・工学研究科, 教授 (10161689)
Co-Investigator(Kenkyū-buntansha)	津本浩平東北大学, 大学院・工学研究科, 助手 (90271866)
Keywords	抗原抗体相互作用 / キメラ受容体 / 分子認識 / 細胞増殖 / 融合蛋白質 / 構造解析
Research Abstract	研究代表者らが展開してきた、抗ニワトリリゾチーム抗体HyHEL10可変領域(Fv,VHとVLからなる)と抗原の相互作用の精密解析、抗原認識能の変換や抗原抗体複合体の構造解析研究と、二量体形成により活性化されるマウスエリスロポエチン受容体とHyHEL10を融合させたキメラ受容体を用いた、細胞増殖の人工的制御技術の開発研究を踏まえ、本研究では(1)HyHEL10Fv-キメラ受容体を用いた抗体分子認識能と細胞増殖制御の解析、(2)HyHEL10の分子認識能の変換による任意のリガンド分子による細胞増殖制御系の構築を目標とし、本年度は研究推進の基盤として、以下の研究を遂行した。 (1)Fv断片の結晶構造解析:HyHEL10野生型及び部位特異的変異導入を施した抗体Fv断片と抗原の複合体について、結晶構造解析を行った。野生型Fvについては、Fabと異なる分子機構で、抗原を認識している可能性が示唆された。塩橋に関与するアミノ酸残基に変異を導入した抗体(32A,96A〉と抗原の複合体については、変異導入の効果が、構造変化による場合(32A)と水分子の導入による場合(96A)が観察された。(2)キメラ受容体:変異を導入した抗体を融合させた受容体蛋白質の大腸菌を用いた発現系の構築を目指した。融合させる、機能変換を施した抗体断片の抗原認識能を詳細に解析し、Fv断片と一本鎖化したscFvの間に認識能の相違が観察された。

Research Products
(21 results)

All Other

All Publications (21 results)

[Publications] Tsumoto et al.: "Novel selection method for engineered artibodies using the mechanism of Fv fragment stabilization in the presenese of antigen" Protein Engineering. 10. 1311-1318 (1997)
[Publications] Furuta et al.: "Constructin of mono-and bi-valent human single-chain Fv fragments against the D-antigen in the Rh blood group" Protein Engineering. 11. 233-241 (1998)
[Publications] Tsumoto et al.: "Highly efficient recovery of functional single-chain Fv fragment from inclusion bodies overexpressed in E coliby contyolled・・・・・・" Journal of Immunological Methods. 219. 119-129 (1998)
[Publications] Koshiba et al.: "Thermal study of mutant human lysozymes with partially introduced Ca^<2+> binding sites by using efficient refolding system" Protein Engineering. 11. 583-590 (1998)
[Publications] Chaudhuri et al.: "Effect of the extva N-terminal methionine vesidue on the stability and folding of recomblnant α-lactalabumin in E.coli" Journal of Molecular Biology. 285. 1179-1199 (1999)
[Publications] Merk et al.: "Cell-free expression of two single-chain monoclonal antibodies against lysozyme" Journal of Biochemistry. 125. 328-331 (1999)
[Publications] 前仲ら: "Structural analysis of mutant hen egg-white lysozyme prefering a minor binding mode" Biochim.Biophys.Acta. 1384. 23-31 (1998)
[Publications] Kitamura et al.: "Cloning and expression of the gene encoding Havodoxin from Desulfovibrio vulgaris (Miyazaki F)" Journal of Biochemistry. 123. 891-898 (1998)
[Publications] Maenaka et al.: "Disclosure of another carbohydrate binding site in hen egg-white lysozyme by conversion of subsite B stmctune" Biochemical Journal. 331. 71-76 (1998)
[Publications] Hori et al.: "Substrate Recognition of tRNA(Guanosine-2′-)-wethyltransferase from Thermus thermophilus HB27" Journal of Biological Chemistry. 273. 25721-25727 (1998)
[Publications] Tanaka et al.: "Pl-Speaficity of Aquualysin I(a Subtilisin-type Serine Protease)from Thermus aquaticus YT-1,using Pl-Substituted Derivatives of SSI" Bioscl.Biotechnol.Biochem.62. 2035-2038 (1998)
[Publications] Sakurai et al.: "SEA-scFv as bifunctional antibody" Biochemical Biophysical Research Communication. 印刷中. (1999)
[Publications] Suzuki et al.: "Open sand wich ELISA with VH-/VL-al kaline phosphatase fusion proteins" Journal of Immunological Methods. 印刷中. (1999)
[Publications] Kitamura et al.: "Expression of a Model Peptide of a Marine Mussel Adhesivo Protein in E.coli." Journal of Polymer Science,A,Polymer Chemistry. 印刷中. (1999)
[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)
[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)
[Publications] 上田ら: "新たな免疫測定法" 遺伝子医学. 3. 170-175 (1999)
[Publications] 熊谷,津本,前仲: "構造生物学(共著)" 朝倉出版, 13 (1998)
[Publications] 熊谷,津本: "遺伝子工学(分子遺伝学共著)" 丸善出版社, 25 (1997)
[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)
[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)

1998 Fiscal Year Annual Research Report

新機能抗体-受容体キメラ蛋白質の創製と細胞増殖制御機構解析への応用

Principal Investigator

熊谷 泉 東北大学, 大学院・工学研究科, 教授 (10161689)

Research Products

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

[Publications] Furuta et al.: "Constructin of mono-and bi-valent human single-chain Fv fragments against the D-antigen in the Rh blood group" Protein Engineering. 11. 233-241 (1998)

[Publications] Tsumoto et al.: "Highly efficient recovery of functional single-chain Fv fragment from inclusion bodies overexpressed in E coliby contyolled・・・・・・" Journal of Immunological Methods. 219. 119-129 (1998)

[Publications] Koshiba et al.: "Thermal study of mutant human lysozymes with partially introduced Ca^<2+> binding sites by using efficient refolding system" Protein Engineering. 11. 583-590 (1998)

[Publications] Chaudhuri et al.: "Effect of the extva N-terminal methionine vesidue on the stability and folding of recomblnant α-lactalabumin in E.coli" Journal of Molecular Biology. 285. 1179-1199 (1999)

[Publications] Merk et al.: "Cell-free expression of two single-chain monoclonal antibodies against lysozyme" Journal of Biochemistry. 125. 328-331 (1999)

[Publications] 前仲ら: "Structural analysis of mutant hen egg-white lysozyme prefering a minor binding mode" Biochim.Biophys.Acta. 1384. 23-31 (1998)

[Publications] Kitamura et al.: "Cloning and expression of the gene encoding Havodoxin from Desulfovibrio vulgaris (Miyazaki F)" Journal of Biochemistry. 123. 891-898 (1998)

[Publications] Maenaka et al.: "Disclosure of another carbohydrate binding site in hen egg-white lysozyme by conversion of subsite B stmctune" Biochemical Journal. 331. 71-76 (1998)

[Publications] Hori et al.: "Substrate Recognition of tRNA(Guanosine-2′-)-wethyltransferase from Thermus thermophilus HB27" Journal of Biological Chemistry. 273. 25721-25727 (1998)

[Publications] Tanaka et al.: "Pl-Speaficity of Aquualysin I(a Subtilisin-type Serine Protease)from Thermus aquaticus YT-1,using Pl-Substituted Derivatives of SSI" Bioscl.Biotechnol.Biochem.62. 2035-2038 (1998)

[Publications] Sakurai et al.: "SEA-scFv as bifunctional antibody" Biochemical Biophysical Research Communication. 印刷中. (1999)

[Publications] Suzuki et al.: "Open sand wich ELISA with VH-/VL-al kaline phosphatase fusion proteins" Journal of Immunological Methods. 印刷中. (1999)

[Publications] Kitamura et al.: "Expression of a Model Peptide of a Marine Mussel Adhesivo Protein in E.coli." Journal of Polymer Science,A,Polymer Chemistry. 印刷中. (1999)

[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)

[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)

[Publications] 上田ら: "新たな免疫測定法" 遺伝子医学. 3. 170-175 (1999)

[Publications] 熊谷,津本,前仲: "構造生物学(共著)" 朝倉出版, 13 (1998)

[Publications] 熊谷,津本: "遺伝子工学(分子遺伝学 共著)" 丸善出版社, 25 (1997)

[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)

[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)

熊谷泉東北大学, 大学院・工学研究科, 教授 (10161689)

[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)

[Publications] 熊谷,津本: "遺伝子工学(分子遺伝学共著)" 丸善出版社, 25 (1997)