2001 Fiscal Year Annual Research Report

構造情報に基づく細胞性免疫システムにおける特異的分子認識機構の解明

Research Project

Project/Area Number	13033002
Research Category	Grant-in-Aid for Scientific Research on Priority Areas (A)
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, 講師 (90271866)
Keywords	T細胞受容体 / ヒト白血球抗原 / X線結晶構造解析 / 巻き戻し系 / 分子認識 / 熱力学的解析 / 不溶性顆粒 / 蛋白質間相互作用
Research Abstract	細胞表面には様々なレセプターが発現し、細胞間の情報伝達に関与している。特に神経系や免疫系に関わるレセプターは様々な疾患に直接関与することから医学的に幅広く注目されている。細胞性免疫を司るT細胞に関しては、マクロファージによって分解された外来蛋白質のフラグメントを付着したヒト白血球抗原(HLA,主要組織適合遺伝子複合体(MHC)とも呼ぶ)がT細胞受容体(TCR)によって認識され、T細胞が活性化されることが知られている。本研究は、このような細胞性免疫において鍵となる相互作用である、TCR-HLA相互作用について、特にTCR-HLAクラスI相互作用に焦点を絞り、分子認識データを立体構造に基づいて収集し、その分子認識能における特異性・親和性を創出する分子機構を明らかにすることを目的としている。本年度の研究成果は以下のとおりである。ヒトT細胞受容体(TCR)とHLAの大量発現系:既にクローニングしてあるヒトTCRとHLAクラスIの細胞外ドメインをコードする遺伝子を用いた、大腸菌で不溶性頼粒として発現させる系を改良したほか希釈法による巻き戻し系を整備した。さらに、すでに研究代表者らが実績のあるイムノグロブリンフォールドの効率よい巻き戻し系をもとに、さらに効率のよいシステムを確立することができた。表面プラスモン共鳴と等温滴定型熱量測定により、その相互作用を精密に解析することができた。 TCR、HLAならびにその複合体の結晶化とX線結晶構造解析:大量調製したTCRとHLAクラスIについて、またその複合体の結晶化を目指した。報告例をもとに、さらによい結晶化条件を探る。次年度における実験室レベルの結晶構造解析のための基盤を作ることができた。

Research Products
(21 results)

All Other

All Publications (21 results)

[Publications] Yoda et al.: "Folding-unfolding of goat aipha-lactalbumin studied by stopped-flow CD and molecular dynamic simulations"Proteins ; Struct. Funct. Genet.. 42. 49-65 (2001)
[Publications] Arai et al.: "Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer"Analytical Biochemistry. 289. 77-81 (2001)
[Publications] Nakamura et al.: "Panning of phage VH library using nitrocellulose membranes -application to selection of human VH library"The Journal of Biochemistry. 129. 209-212 (2001)
[Publications] Kasai et al.: "Fv fragment of an antibody specific for hepatitis C NS3 protease domain : The sequence analysis, bacterial expression and functional characterization"Biochem. Biophys. Res. Commun.. 281. 416-424 (2001)
[Publications] Ueno et al.: "T-cell Immunotherapy for Human MK-1-Expressing Tumors Using a Fusion Protein of the Superantigen SEA and Anti-MK-1 scFv Antibody"Anticancer research. 22(印刷中). (2002)
[Publications] 津本: "不溶性顆粒からの蛋白質の巻き戻し"蛋白質核酸酵素. 46. 1238-1246 (2001)
[Publications] 津本, 熊谷: "ファージディスプレーを用いた新規バイオ分子の選択と応用"BLOINDUSTRY. 18. 26-33 (2001)
[Publications] Nakamra et al.: "Detection of biotinylated proteins in polyacrylamide gels using an avidin-fluorescein conjugate"Analytical Biochemistry. (印刷中). (2002)
[Publications] Shiroishi et al.: "Structural evidence for entropic contribution of salt bridge formation to a protein antigen-antibody interaction : The case of HyHEL-10 Fv-hen lysozyme complex"The Journal of Biological Chemistry. 276. 23042-23050 (2001)
[Publications] Kawahara et al.: "Replacing factor dependency with that for lysozyme : Affordable culture of IL-6 dependent hybridoma by transfecting artificial cell surface receptor"Biotechnology and Bioengineering. 76. 416-423 (2001)
[Publications] Nakamura et al.: "The effect of an agglutogen on virus infection : biotinylated filamentous phages and avidin as a model"FEBS Letters. (印刷中). (2002)
[Publications] Kawahara et al.: "Selection of highly productive mammalian cells based on an inducible growth advantage using an antibody/receptor chimera"J. Biosci. Biotechnol.. (印刷中). (2002)
[Publications] Kawahara et al.: "A growth signal with an artificially induced erythropoietin receptor-gp 130 cytoplasmic domain heterodimer"The Journal of Biochemistry. 130. 305-312 (2001)
[Publications] Horii et al.: "Contribution of Thr29 to the Thermodynamic Stability of Goat α-Lactalbumin As Determined by Experimental and Theoretical Approaches"PROTEINS : Struct. Funct. Genet.. 45. 16-29 (2001)
[Publications] Nakamura et al.: "A visualization method of filamentous phage infection and phage-derived proteins in Escherichia coil using biotinylated phages"Biochem. Biophys. Res. Commun.. 289. 252-256 (2001)
[Publications] Kodama et al.: "Mutated SEA D227A-conjugated antibodies greatly enhance antitumor activity against MUC1-expressing bile duct carcinoma (BDC)"Cancer Immunology Immunotherapy. 50. 539-548 (2001)
[Publications] Nakamura et al.: "Phage library panning against cytosolic fraction of cells using quantitative dot blotting : Application of selected VH to histochemistry"Journal of Immunological Methods. 261. 65-72 (2002)
[Publications] Takemura et al.: "A mutated superantigen SEA D227A fusion diabody specific to MUC1 and CD3 for targeted cancer immunotherapy for bile duct carcinoma"Cancer Immunology Immunotherapy. (印刷中). (2002)
[Publications] Kumagai, Tsumoto: "Encyclopedia of Life Science"Blackwell(印刷中). (2002)
[Publications] 津本, 熊谷: "コンビナトリアルバイオエンジニアリング"化学同人(印刷中). (2002)
[Publications] 津本, 熊谷: "新タンパク質科学実験法第3巻構造機能解析"東京化学同人(印刷中). (2002)

2001 Fiscal Year Annual Research Report

構造情報に基づく細胞性免疫システムにおける特異的分子認識機構の解明

Principal Investigator

熊谷 泉 東北大学, 大学院・工学研究科, 教授 (10161689)

Research Products

[Publications] Yoda et al.: "Folding-unfolding of goat aipha-lactalbumin studied by stopped-flow CD and molecular dynamic simulations"Proteins ; Struct. Funct. Genet.. 42. 49-65 (2001)

[Publications] Arai et al.: "Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer"Analytical Biochemistry. 289. 77-81 (2001)

[Publications] Nakamura et al.: "Panning of phage VH library using nitrocellulose membranes -application to selection of human VH library"The Journal of Biochemistry. 129. 209-212 (2001)

[Publications] Kasai et al.: "Fv fragment of an antibody specific for hepatitis C NS3 protease domain : The sequence analysis, bacterial expression and functional characterization"Biochem. Biophys. Res. Commun.. 281. 416-424 (2001)

[Publications] Ueno et al.: "T-cell Immunotherapy for Human MK-1-Expressing Tumors Using a Fusion Protein of the Superantigen SEA and Anti-MK-1 scFv Antibody"Anticancer research. 22(印刷中). (2002)

[Publications] 津本: "不溶性顆粒からの蛋白質の巻き戻し"蛋白質核酸酵素. 46. 1238-1246 (2001)

[Publications] 津本, 熊谷: "ファージディスプレーを用いた新規バイオ分子の選択と応用"BLOINDUSTRY. 18. 26-33 (2001)

[Publications] Nakamra et al.: "Detection of biotinylated proteins in polyacrylamide gels using an avidin-fluorescein conjugate"Analytical Biochemistry. (印刷中). (2002)

[Publications] Shiroishi et al.: "Structural evidence for entropic contribution of salt bridge formation to a protein antigen-antibody interaction : The case of HyHEL-10 Fv-hen lysozyme complex"The Journal of Biological Chemistry. 276. 23042-23050 (2001)

[Publications] Kawahara et al.: "Replacing factor dependency with that for lysozyme : Affordable culture of IL-6 dependent hybridoma by transfecting artificial cell surface receptor"Biotechnology and Bioengineering. 76. 416-423 (2001)

[Publications] Nakamura et al.: "The effect of an agglutogen on virus infection : biotinylated filamentous phages and avidin as a model"FEBS Letters. (印刷中). (2002)

[Publications] Kawahara et al.: "Selection of highly productive mammalian cells based on an inducible growth advantage using an antibody/receptor chimera"J. Biosci. Biotechnol.. (印刷中). (2002)

[Publications] Kawahara et al.: "A growth signal with an artificially induced erythropoietin receptor-gp 130 cytoplasmic domain heterodimer"The Journal of Biochemistry. 130. 305-312 (2001)

[Publications] Horii et al.: "Contribution of Thr29 to the Thermodynamic Stability of Goat α-Lactalbumin As Determined by Experimental and Theoretical Approaches"PROTEINS : Struct. Funct. Genet.. 45. 16-29 (2001)

[Publications] Nakamura et al.: "A visualization method of filamentous phage infection and phage-derived proteins in Escherichia coil using biotinylated phages"Biochem. Biophys. Res. Commun.. 289. 252-256 (2001)

[Publications] Kodama et al.: "Mutated SEA D227A-conjugated antibodies greatly enhance antitumor activity against MUC1-expressing bile duct carcinoma (BDC)"Cancer Immunology Immunotherapy. 50. 539-548 (2001)

[Publications] Nakamura et al.: "Phage library panning against cytosolic fraction of cells using quantitative dot blotting : Application of selected VH to histochemistry"Journal of Immunological Methods. 261. 65-72 (2002)

[Publications] Takemura et al.: "A mutated superantigen SEA D227A fusion diabody specific to MUC1 and CD3 for targeted cancer immunotherapy for bile duct carcinoma"Cancer Immunology Immunotherapy. (印刷中). (2002)

[Publications] Kumagai, Tsumoto: "Encyclopedia of Life Science"Blackwell(印刷中). (2002)

[Publications] 津本, 熊谷: "コンビナトリアルバイオエンジニアリング"化学同人(印刷中). (2002)

[Publications] 津本, 熊谷: "新タンパク質科学実験法 第3巻構造機能解析"東京化学同人(印刷中). (2002)

熊谷泉東北大学, 大学院・工学研究科, 教授 (10161689)

[Publications] 津本, 熊谷: "新タンパク質科学実験法第3巻構造機能解析"東京化学同人(印刷中). (2002)