タンパク質工学の応用へ向けての多角的アプローチ

1994 Fiscal Year Annual Research Report

• Project/Area Number: 06304015
• Research Institution: Kyoto University
• Principal Investigator: 外村 辨一郎 京都大学, 農学部, 教授 (20026545)
• Co-Investigator(Kenkyū-buntansha): 崎山 文夫 大阪大学, 蛋白質研究所, 教授 (40029947) ; 小田 順一 京都大学, 化学研究所, 教授 (50027041) ; 岩永 貞昭 九州大学, 理学部, 教授 (90029942) ; 井本 泰治 九州大学, 薬学部, 教授 (90038282) ; 芦田 玉一 名古屋大学, 工学部, 教授 (10029936)
• Keywords: タンパク質工学 / タンパク質の化学修飾 / タンパク質の耐熱性 / タンパク質の構造 / 酵素の基質特異性 / タンパク質間特異的分子認識 / 遺伝子の塩基配列 / 部位特異的変異

Research Abstract

蛋白質性インヒビターとプロテアーゼとの複合体及びリパーゼA2のX線結晶構造解析により、詳細な構造情報を得た(芦田)。酵素の耐熱化を目指して、リゾチームの高温不可逆失活の機構および還元条件からの構造形成の過程を解析した(井本)。血液凝固系において、組織因子(TF)上のVIIa因子結合部位の解析を行い、TFのLys-17の結合への関与を明らかにした(岩永)。ヘビ毒腺ホスホリパーゼA2の59位及び31位のアミノ酸を置換し、それが基質との相互作用や四次構造に及ぼす効果を解析した(大野)。大腸菌由来グルタチオン合成酵素のATP結合部位の17残基からなるループ構造が担う機能を、ループ欠損変異体酵素を作成して解析した(小田)。グルタチオン合成酵素のポリエチレングリコールによる化学修飾により、安定性増大とアナフィラキシーショック低減がもたらされた(木村)。^<15>Nラベルの1本鎖モネリンの構造を多次元NMR法により解析し、詳細な構造情報を得た(小林)。アクロモバクター・プロテアーゼの部位特異的変異酵素を用いて、活性および基質特異性への水素結合の寄与を評価した(山崎)。Bacillus由来オリゴ-1,6-グルコシダーゼにプロリン残基9個を導入し、酵素の耐熱性の累積的増大を明らかにし、耐熱性プロリン説の直接証明に成功した(鈴木)。Bacillus由来リジルtRNA合成酵素の遺伝子の塩基配列を解析し、また酵素のリジン基質認識機構を検討した(外村)。2種類の芳香族化合物酸素添加酵素の各サブユニットを交換したハイブリッド酵素を構築し、環境汚染物質であるトリクロロエチレンをも速やかに分解する酵素を得た(古川)。蛋白質安定化のための修飾剤として新規なデキストラン誘導体を開発し、その有効性を示した(山崎)。4種のプロリルアミノペプチダーゼの遺伝子クローニング、塩基配列解析、酵素過剰発現、及び結晶化に成功した。本酵素がセリン酵素であることを解明した(芳本)。

Research Products

• [Publications] Suzuki,A.: "Crystal Structure Analysis of phospholipase A2 from..." Journal of Biochemistry. 117(in press). (1995)
• [Publications] Yamane,T.: "Structure of a New Alkaline Serine Protease..." Acta Crystallographica. D51(in press). (1995)
• [Publications] Tomizawa,H.: "The Mechanism of Irreversidle Inactivation of Lysozyme." Biochemistry. 33. 13032-13037 (1994)
• [Publications] Tomizawa,H.: "Isolation and Charecterization of 101-Succinimide..." Biochemistry. 33. 8770-8774 (1994)
• [Publications] Maeda,Y.: "The Role of Net Charge on the Renaturation of Redyced..." Protein Engineering. 7. 1249-1254 (1994)
• [Publications] Imoto,T.: "Lysozyme Requires Fluctuation of the Active Site for..." Protein Engineering. 7. 743-748 (1994)
• [Publications] Ueda,T.: "Formation of alpha-Helix 88-98 is Essential in the .." Journal of Molecular Biology. 235. 1312-1317 (1994)
• [Publications] Iwanaga,S.: "A Role of Hemocyte-derived Granular Components in..." Annales of New York Academy of Sciences. 712. 102-116 (1994)
• [Publications] Higashi,S.: "Identification of Regions of Bovine Factor VII..." Journal of Biological Chemistry. 269. 18891-18898 (1994)
• [Publications] Miura,Y.: "A Horseshoe Crab Intercelluar Coagulation Inhibitor..." Journal of Biological Chemistry. 269. 542-547 (1994)
• [Publications] Seki,N.: "Horseshoe Crab (1,3)-beta-D-Glucan-sensitive....." Journal of Biological Chemistry. 269. 1370-1374 (1994)
• [Publications] Nishda,S.: "cDNA Cloning and Deduced Amino Acid Sequence of..." Biochemistry. 34. 1771-1778 (1995)
• [Publications] Kishan,L.A.: "Activation Peptide of Human Factor IX has..." Biochemistry. 33. 5167-5171 (1994)
• [Publications] Alam,R.: "cDNA Cloning and Characterization of Mitochondrial.." Journal of Biochemistry. 116. 416-425 (1994)
• [Publications] Yoshida,M.: "D-myo-Inositol 1,4,5-Triphosphate Binding Protein.." Journal of Biochemistry. 115. 973-980 (1994)
• [Publications] Muta,T.: "Purified Horseshoe Crab Factor G:Reconstitution and.." Journal of Biological Chemistry. 270. 892-897 (1995)
• [Publications] Miura,Y.: "Limulus Intracellular Coagulation Inhibitor Type 2:.." Journal of Biological Chemistry. 270. 558-565 (1995)
• [Publications] Kato,T.: "Interaction of N-Terminal Fragments of Group I and..." Biochemica et Biophysica Acta. 1211. 215-220 (1994)
• [Publications] Nose,T.: "Purification and Characterization of a Coagulant..." Toxicon. 22. 1509-1520 (1994)
• [Publications] Nakashima,K.: "Polymorphisms of Trimeresurus flaviridis Venom Gland.." Bioscience,Biotechnology,& Biochemistry. 58. 1510-1511 (1994)
• [Publications] Nakashima,K.: "Structures of Genes Encoding TATA Box-Binding Proteins.." Gene. 152. 209-213 (1995)
• [Publications] Nakashima,K.: "Accelerated Evolution in the Protein-Coding Regions.." Proc.Natl.Acad.Sci.USA. 92(in press). (1995)
• [Publications] Kato,H.: "Flexible Loop that is a Novel Catalytic Machinery.." Biochemistry. 33. 4995-4999 (1994)
• [Publications] Norioka,S.: "Identification of Three Catalytic Triad Constituents.." Journal of Biochemistry. 269. 17025-17029 (1994)
• [Publications] Watanabe,K.: "Multiple Proline Substitutions Cumulatively Thermo-..." European Journal of Biochemistry. 226. 277-283 (1994)
• [Publications] Inouye,K.: "Dissociation of Dimer of Bovine Erythrocyte Cu,Zn-..." Journal of Biochemistry. 115. 507-515 (1994)
• [Publications] Inouye,K.: "A Spectrophotometric Study on the Interaction of.." Journal of Biochemistry. 116. 530-535 (1994)
• [Publications] Tanaka,F.: "Analysis of Internal Motion of Single Tryptophan..." Biophysical Journal. 67. 874-880 (1994)
• [Publications] Yamashita,H.: "Effect of a p-Nitro Group of Phenyl-maltooligo-..." Biochem.Molecular Biol.International. 35. 79-85 (1995)
• [Publications] Furukawa,K.: "Efficient Degradation of Trichloroethylene by..." Journal of Bacteriology. 176. 2121-2123 (1994)
• [Publications] Hirose,J.: "Construction of Hybrid Biphenyl(bph) and Toluene(tod).." Gene. 138. 27-33 (1994)
• [Publications] Hirose,J.: "Functional and Structural Relationship of Various.." FEMS Microbiology Letters. 118. 273-278 (1994)
• [Publications] Kawamura,S.: "Cloning of the Gene Encoding DNA Binding Protein HU..." Bioscience,Biotechnology,& Biochemistry. 59. 126-129 (1995)
• [Publications] Hatakeyama,T.: "Interaction of the Hemolytic Lectin CEL-III from..." Journal of Biological Chemistry. 270. 3560-3564 (1995)
• [Publications] Hatakeyama,T.: "Purification and Characterization of Four Ca^<++>-..." Journal of Biochemistry. 116. 209-214 (1994)
• [Publications] Ito,K.: "Cloning and High-level Expression of the Glutathione-.." Journal of Bacterilogy. 176. 2483-2491 (1994)
• [Publications] Yoshimoto,T.: "Substrate Specificity of Aminopeptidase P from E.coli" Archives of Biochemistry and Biopysics. 311. 28-34 (1994)
• [Publications] Fukunari,A.: "Colocalization of Prolyl Endopeptidase and Amyloid.." Neuroscience Letter. 176. 201-204 (1994)
• [Publications] Kitazono,A.: "Isolation and Characterization of the Prolyl Amino-.." Journal of Biochemistry. 116. 818-825 (1994)
• [Publications] Kitazono,A.: "Prolyl Aminopeptidase is Not a Sulfhydryl Enzyme:.." Journal of Biochemistry. 116. 943-945 (1994)
• [Publications] Takeya,H.: "Biological Functions of Proteases snd Inhibitors" Japan Scientific Societies Press,Tokyo, 231-252 (1994)
• [Publications] Iwanaga,S.: "Phylogenetic Perspectives in Immunity:The Insect.." R.G.Landes Co.Biomedical Publishers,Austin, 79-96 (1994)
• [Publications] Iwanaga,S.: "Ciba Foundation Symposium No.186,“Antimicrobial" John Willey & Sons.UK and New York, 79-96 (1994)
• [Publications] 岩永貞昭: "血栓・止血・線溶" 中外医学社, 38- (1994)
• [Publications] 岩永貞昭: "ビタミンK-医学・生物学領域における新展開" メディカルジャーナル社, 495- (1994)
• [Publications] Sakiyama,F.: "Methods in Enzymology" Academic Press,New York,(volum 244), 126-137 (1994)
• [Publications] Tsuru,D.: "Methods in Enzymology" Academic Press,New York,(volum 244), 201-215 (1994)