1995 年度実績報告書

タンパク質工学の応用へ向けての多角的アプローチ

研究課題

研究課題/領域番号	06304015
研究機関	京都大学
研究代表者	外村辨一郎京都大学, 農学部, 教授 (20026545)
研究分担者	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)
キーワード	タンパク質工学 / タンパク質のX線結晶解析 / タンパク質の構造 / タンパク質の耐熱安定性 / 遺伝子の塩基配列 / 酵素の基質特異性 / タンパク質の化学修飾 / タンパク質のNMR
研究概要	小麦胚芽トリプシンインヒビターの単体とトリプシンの複合体の立体構造をX線結晶構造解析により解明した(芦田)。組換え型ニワトリ卵白リゾチームを用いて酵素反応機構をカルボニウムイオン中間体安定化機構からグリコシル中間体経由機構への変換に成功した(井本)。カブトガニ体液の凝固に関与するセリンプロテアーゼの前駆体の1,3-β-D-グルカンによる活性化機構を解明した(岩永)。ハブ属毒腺ホスホリパーゼA2の11種のアイソザイムを単離し一次構造と各遺伝子の塩素配列を解明し進化論的考察を加えた(大野)。グルタチオン合成酵素と遷移状態アナログとの複合体並びにアスパラギンの酸合成酵素の立体構造をX線結晶解析により明らかにした(小田)。臨床治療への利用を目的として、微生物由来のプリンヌクレオシド代謝系酵素の生化学性質並びに酵素遺伝子の塩基配列を解明した(木村)。1本鎖モネリンの溶液構造を多次元NMR法により詳細に解析した(小林)。Achromobacterプロテアーゼ1のリジン残基に対する高い基質特異性の原因をタンパク質工学的に改変した酵素を用いて解明した(崎山)。耐熱性菌のオリゴ-1,6-グルコシダーゼを対象として、プロリン残基の多重置換導入による酵素の累積的耐熱化をタンパク質工学的手法により実験的に証明した(鈴木)。B. stearothermophilusのリジルtRNA合成酵素と基質し-リジンとATPの結合を平衡論的及び速度論的に解析し酵素遺伝子の塩基配列を解明した(外村)。基質特異性の異なる2種のジオキシゲナーゼ間のハイブリッド酵素を作成し芳香族基質に対する特異性の拡大を証明した(古川)。メトキシPEG誘導体によるリゾチームの修飾により酵素の安定性とグラム陰性菌に対する反応性の増大を見出した(山崎)。プロリン残基に特異性を有する微生物由来のアミノペプチダーゼPを単離精製し、酵素遺伝子のクローン化、塩基配列の決定、過剰発現並びに酵素の結晶化を行った(芳本)。

研究成果

(52件)

すべてその他

すべて文献書誌 (52件)

[文献書誌] Yamane, T.: "Crystal structure of Straptomyces erythraeus trypsin..." Journal of Biochemistry. 118. 882-894 (1995)
[文献書誌] Maeda, Y.: "Effective renaturation of reduced lysozyme by gentle..." Protein Engineering. 8. 201-205 (1995)
[文献書誌] Tomizawa, H.: "Effects of additives on irreveasible inactivation of..." Journal of Biochemistry. 117. 369-373 (1995)
[文献書誌] Ito, Y.: "Possible digestive fonction based on sequence and enzymatic..." Journal of Biochemistry. 118. 546-551 (1995)
[文献書誌] Tomizawa, H.: "Stabilization of lysozyme against irrevesible inactivation..." Journal of Biochemistry. 117. 635-640 (1995)
[文献書誌] Abe, Y.: "Effect of salt concentration on pKa of acidic residues..." Journal of Biochemistry. 118. 946-952 (1995)
[文献書誌] Motoshima, H.: "Correlationship between the diffrence of energy..." Journal of Biochemistry. 118. 1138-1144 (1995)
[文献書誌] Tomizawa, H.: "Stabilization of lysozyme against irreversible inactivation..." Protein Engineering. 8. 1023-1028 (1995)
[文献書誌] Ueda, T.: "Kinetically trapped structure in the renaturation of..." Biochemistry. 34. 16178-16185 (1995)
[文献書誌] Ueda, T.: "Stabilization of lysozyme by introducing-N-glycosylation..." Journal of Biochemistry. 119. 157-161 (1996)
[文献書誌] Hashimoto, Y.: "A mutation study of the catalytic residue Asp52 in hen egg..." Journal of Biochemistry. 119. 145-150 (1996)
[文献書誌] Saito, T.: "A novel big defensin foud in horse-shoe crab hemocytes:..." Journal of Biochemistry. 117. 1131-1137 (1995)
[文献書誌] Saito, T.: "A novel type of limulus lectin-L6 : purification, covalent..." Journal of Biological Chemistry. 270. 14493-14499 (1995)
[文献書誌] Okino, N.: "Purification, chracterization and cDNA cloning of a 27-kDa..." Journal of Biological Chmistry. 270. 31008-31015 (1995)
[文献書誌] Lal Agawara, K.: "A cysteine protease inhibitor stored in the large granules..." Journal of Biochemistry. 119. 85-94 (1996)
[文献書誌] Takeuchi, M.: "The localization of lipopolysaccharide in an endotoxemic..." Path. Res. Pract.190. 1123-1133 (1995)
[文献書誌] Kumasaka, T.: "Crystallization and preliminary X-ray study H_2-proteinase" Journal of Biochemistry. 117. 929-930 (1995)
[文献書誌] Fujimoto, K.: "Nucleotide sequence of the cDNA encoding prophenoloxidase..." Pro. Natl. Acad. Sci. USA. 92. 7769-7773 (1995)
[文献書誌] Kojima, N.: "Substrate specificity of rabbit liver metalloendopeptidase..." Journal of Biochemistry. 118. 855-861 (1995)
[文献書誌] Kumasaka, T.: "Crystal structure of H_2-proteinase from the venom of..." Journal of Biochemistry. 119. 49-57 (1996)
[文献書誌] Ogawa, T.: "Trimeresurus flavoviridis venom phospholipase A_2 genes..." Journal of Molecular Recognition. 8. 40-46 (1995)
[文献書誌] Shimohigashi, Y.: "Human thrombin receptors are insensitive to thrombin-like..." Biochem. Mol. Biol. Int.35. 415-421 (1995)
[文献書誌] Nakai, M.: "Purification and primary structure of a mutoxic lysine-49..." Toxicon. 33. 1469-1478 (1995)
[文献書誌] Shimohigashi, Y.: "Lysine-49-phospholipase A_2 from Trimeresurus flavoviridis..." Journal of Biochemistry. 118. 1037-1044 (1995)
[文献書誌] Ogawa, T.: "Localization and expression of phospholipases A_2..." Toxicon. 33. 1645-1652 (1995)
[文献書誌] Ogawa, T.: "Molecular evolution of group II phospholipases A_2." Journal of Molecular Evolution. (in press). (1996)
[文献書誌] Hara, T.: "Site-directed mmutagenesis of glutathione synthetase..." Protein Engineering. 8. 711-716 (1995)
[文献書誌] Hibi, T.: "Affinity labelling as a tool for fixing the flexible..." Nature Structural Biology. 3. 16-18 (1996)
[文献書誌] Nakatsu, T.: "Crystallization and preliminary crystallographic study..." Acta Crystallographica D. (in press). (1996)
[文献書誌] Takehara, M.: "Molecular cloning and nucleotide sequence of purine..." Biosci. Biothch. Biochem.59. 1987-1990 (1995)
[文献書誌] Kaktahira, R.: "Solution structure of a human calcitonin analog elucidated..." Int. J. Peptide Protein Res.45. 305-311 (1995)
[文献書誌] Nemoto, N.: "Solution structure of w-conotoxin MVIIC datermined by NMR." Biochem. Biophys. Res. Commun.207. 695-700 (1995)
[文献書誌] Nemoto, N.: "Solution structure of adrenocorticotropic hormone fragment..." Peptide Chemistry. 1994. 81-84 (1995)
[文献書誌] Aumelas, A.: "〔Lys-2-Arg-1〕-endothelin-1 solution structure by 2D-H-NMR;" Biochemistry. 34. 4546-4561 (1995)
[文献書誌] Uegaki, K.: "15N labeling method of peptides using a thioredoxin gene..." FEBS Letters. 379. 47-50 (1996)
[文献書誌] 鈴木譲: "タンパク質の高温適応とプロリン残基の役割" 生化学. 67. 455-458 (1995)
[文献書誌] Takii, Y.: "B. stearothermophilus ATCC 12016 1-glucosidase specific..." Appl. Microbiol. Biotechnol.(in press). (1996)
[文献書誌] Takita, T.: "Lysine-tRNA synthetase from B. stearothermophilus..." Journal of Biochemistry. 119(inpress). (1996)
[文献書誌] Furukawa, K.: "Biochemistry and genetics of PCB metabolism." Environ. Health Perspectives. 103. 21-23 (1995)
[文献書誌] Kimura, N.: "Analysis of substrate range of biphenyl-catabolic enzymes..." Biosci. Biotech. Biochem.60. 220-223 (1995)
[文献書誌] Yamashita, S.: "The steady state and time-resolved flourescence studies on..." Biosci. Biotech. Biochem.59. 1255-1261 (1995)
[文献書誌] Yamashita, S.: "Segmental motion of subsite C of henn egg-white lysozyme:" Biosci. Biotech. Biochem.59. 1579-1580 (1995)
[文献書誌] Ohba, H.: "Improvement of the thermostability of pyruvate decarboxylase..." Biosci. Biotech. Biochem.59. 1581-1583 (1995)
[文献書誌] Suga, K.: "Prolyl aminopeptidase (Angiotensinase C) : Purification..." Biosci. Biotech. Biochem.59. 298-301 (1995)
[文献書誌] Yoshimoto, T.: "Protease II from Moraxella lacunata. Cloning, seqencing,..." Journal of Biochemistry. 117. 654-660 (1995)
[文献書誌] Kabashima, T.: "Dipeptidyl aminopeptidase IV from Flavobacterium." Arch. Biochim. Biophys.320. 123-128 (1995)
[文献書誌] Fujita, Y.: "Partial purification and characterization of an esterase..." Biol. Pharm. Bull.18. 648-652 (1995)
[文献書誌] Suga, K.: "Prolidase from Xanthomonas maltophilia : Purification and..." Bikosci.Biotech. Biochem.59. 2087-2090 (1995)
[文献書誌] Muta, T.: "Progress in Molecular & Subcellular Biology vol.15 (in press)" Springer-Verlag, Berlin, (1996)
[文献書誌] Kawabata, S.: "New Directions in Invertebrate Immunology (in press)" SOS Publications, Fair Haven, NJ., (1996)
[文献書誌] Muta, T.: "Current Opinion in Immunology" Current Biology Ltd. London (in press), (1996)
[文献書誌] 山崎信行: "低アレルギー食品の開発と展望(池澤善郎編)" シーエムシー, 79-106 (1995)

1995 年度 実績報告書

タンパク質工学の応用へ向けての多角的アプローチ

研究代表者

外村 辨一郎 京都大学, 農学部, 教授 (20026545)

研究成果

[文献書誌] Yamane, T.: "Crystal structure of Straptomyces erythraeus trypsin..." Journal of Biochemistry. 118. 882-894 (1995)

[文献書誌] Maeda, Y.: "Effective renaturation of reduced lysozyme by gentle..." Protein Engineering. 8. 201-205 (1995)

[文献書誌] Tomizawa, H.: "Effects of additives on irreveasible inactivation of..." Journal of Biochemistry. 117. 369-373 (1995)

[文献書誌] Ito, Y.: "Possible digestive fonction based on sequence and enzymatic..." Journal of Biochemistry. 118. 546-551 (1995)

[文献書誌] Tomizawa, H.: "Stabilization of lysozyme against irrevesible inactivation..." Journal of Biochemistry. 117. 635-640 (1995)

[文献書誌] Abe, Y.: "Effect of salt concentration on pKa of acidic residues..." Journal of Biochemistry. 118. 946-952 (1995)

[文献書誌] Motoshima, H.: "Correlationship between the diffrence of energy..." Journal of Biochemistry. 118. 1138-1144 (1995)

[文献書誌] Tomizawa, H.: "Stabilization of lysozyme against irreversible inactivation..." Protein Engineering. 8. 1023-1028 (1995)

[文献書誌] Ueda, T.: "Kinetically trapped structure in the renaturation of..." Biochemistry. 34. 16178-16185 (1995)

[文献書誌] Ueda, T.: "Stabilization of lysozyme by introducing-N-glycosylation..." Journal of Biochemistry. 119. 157-161 (1996)

[文献書誌] Hashimoto, Y.: "A mutation study of the catalytic residue Asp52 in hen egg..." Journal of Biochemistry. 119. 145-150 (1996)

[文献書誌] Saito, T.: "A novel big defensin foud in horse-shoe crab hemocytes:..." Journal of Biochemistry. 117. 1131-1137 (1995)

[文献書誌] Saito, T.: "A novel type of limulus lectin-L6 : purification, covalent..." Journal of Biological Chemistry. 270. 14493-14499 (1995)

[文献書誌] Okino, N.: "Purification, chracterization and cDNA cloning of a 27-kDa..." Journal of Biological Chmistry. 270. 31008-31015 (1995)

[文献書誌] Lal Agawara, K.: "A cysteine protease inhibitor stored in the large granules..." Journal of Biochemistry. 119. 85-94 (1996)

[文献書誌] Takeuchi, M.: "The localization of lipopolysaccharide in an endotoxemic..." Path. Res. Pract.190. 1123-1133 (1995)

[文献書誌] Kumasaka, T.: "Crystallization and preliminary X-ray study H_2-proteinase" Journal of Biochemistry. 117. 929-930 (1995)

[文献書誌] Fujimoto, K.: "Nucleotide sequence of the cDNA encoding prophenoloxidase..." Pro. Natl. Acad. Sci. USA. 92. 7769-7773 (1995)

[文献書誌] Kojima, N.: "Substrate specificity of rabbit liver metalloendopeptidase..." Journal of Biochemistry. 118. 855-861 (1995)

[文献書誌] Kumasaka, T.: "Crystal structure of H_2-proteinase from the venom of..." Journal of Biochemistry. 119. 49-57 (1996)

[文献書誌] Ogawa, T.: "Trimeresurus flavoviridis venom phospholipase A_2 genes..." Journal of Molecular Recognition. 8. 40-46 (1995)

[文献書誌] Shimohigashi, Y.: "Human thrombin receptors are insensitive to thrombin-like..." Biochem. Mol. Biol. Int.35. 415-421 (1995)

[文献書誌] Nakai, M.: "Purification and primary structure of a mutoxic lysine-49..." Toxicon. 33. 1469-1478 (1995)

[文献書誌] Shimohigashi, Y.: "Lysine-49-phospholipase A_2 from Trimeresurus flavoviridis..." Journal of Biochemistry. 118. 1037-1044 (1995)

[文献書誌] Ogawa, T.: "Localization and expression of phospholipases A_2..." Toxicon. 33. 1645-1652 (1995)

[文献書誌] Ogawa, T.: "Molecular evolution of group II phospholipases A_2." Journal of Molecular Evolution. (in press). (1996)

[文献書誌] Hara, T.: "Site-directed mmutagenesis of glutathione synthetase..." Protein Engineering. 8. 711-716 (1995)

[文献書誌] Hibi, T.: "Affinity labelling as a tool for fixing the flexible..." Nature Structural Biology. 3. 16-18 (1996)

[文献書誌] Nakatsu, T.: "Crystallization and preliminary crystallographic study..." Acta Crystallographica D. (in press). (1996)

[文献書誌] Takehara, M.: "Molecular cloning and nucleotide sequence of purine..." Biosci. Biothch. Biochem.59. 1987-1990 (1995)

[文献書誌] Kaktahira, R.: "Solution structure of a human calcitonin analog elucidated..." Int. J. Peptide Protein Res.45. 305-311 (1995)

[文献書誌] Nemoto, N.: "Solution structure of w-conotoxin MVIIC datermined by NMR." Biochem. Biophys. Res. Commun.207. 695-700 (1995)

[文献書誌] Nemoto, N.: "Solution structure of adrenocorticotropic hormone fragment..." Peptide Chemistry. 1994. 81-84 (1995)

[文献書誌] Aumelas, A.: "〔Lys-2-Arg-1〕-endothelin-1 solution structure by 2D-H-NMR;" Biochemistry. 34. 4546-4561 (1995)

[文献書誌] Uegaki, K.: "15N labeling method of peptides using a thioredoxin gene..." FEBS Letters. 379. 47-50 (1996)

[文献書誌] 鈴木譲: "タンパク質の高温適応とプロリン残基の役割" 生化学. 67. 455-458 (1995)

[文献書誌] Takii, Y.: "B. stearothermophilus ATCC 12016 1-glucosidase specific..." Appl. Microbiol. Biotechnol.(in press). (1996)

[文献書誌] Takita, T.: "Lysine-tRNA synthetase from B. stearothermophilus..." Journal of Biochemistry. 119(inpress). (1996)

[文献書誌] Furukawa, K.: "Biochemistry and genetics of PCB metabolism." Environ. Health Perspectives. 103. 21-23 (1995)

[文献書誌] Kimura, N.: "Analysis of substrate range of biphenyl-catabolic enzymes..." Biosci. Biotech. Biochem.60. 220-223 (1995)

[文献書誌] Yamashita, S.: "The steady state and time-resolved flourescence studies on..." Biosci. Biotech. Biochem.59. 1255-1261 (1995)

[文献書誌] Yamashita, S.: "Segmental motion of subsite C of henn egg-white lysozyme:" Biosci. Biotech. Biochem.59. 1579-1580 (1995)

[文献書誌] Ohba, H.: "Improvement of the thermostability of pyruvate decarboxylase..." Biosci. Biotech. Biochem.59. 1581-1583 (1995)

[文献書誌] Suga, K.: "Prolyl aminopeptidase (Angiotensinase C) : Purification..." Biosci. Biotech. Biochem.59. 298-301 (1995)

[文献書誌] Yoshimoto, T.: "Protease II from Moraxella lacunata. Cloning, seqencing,..." Journal of Biochemistry. 117. 654-660 (1995)

[文献書誌] Kabashima, T.: "Dipeptidyl aminopeptidase IV from Flavobacterium." Arch. Biochim. Biophys.320. 123-128 (1995)

[文献書誌] Fujita, Y.: "Partial purification and characterization of an esterase..." Biol. Pharm. Bull.18. 648-652 (1995)

[文献書誌] Suga, K.: "Prolidase from Xanthomonas maltophilia : Purification and..." Bikosci.Biotech. Biochem.59. 2087-2090 (1995)

[文献書誌] Muta, T.: "Progress in Molecular & Subcellular Biology vol.15 (in press)" Springer-Verlag, Berlin, (1996)

[文献書誌] Kawabata, S.: "New Directions in Invertebrate Immunology (in press)" SOS Publications, Fair Haven, NJ., (1996)

[文献書誌] Muta, T.: "Current Opinion in Immunology" Current Biology Ltd. London (in press), (1996)

[文献書誌] 山崎信行: "低アレルギー食品の開発と展望(池澤善郎編)" シーエムシー, 79-106 (1995)

1995 年度実績報告書

外村辨一郎京都大学, 農学部, 教授 (20026545)