1989 Fiscal Year Annual Research Report

蛋白質工学的手法を活用した徴生物起源の蛋白性プロテア-ゼインヒビタ-の多角的研究

Research Project

Project/Area Number	62300011
Research Institution	Kyoto University
Principal Investigator	廣海啓太郎京都大学, 農学部, 名誉教授 (50025425)
Co-Investigator(Kenkyū-buntansha)	赤坂一之京都大学, 理学部, 助教授 (50025368) 三井幸雄長岡技術科学大学, 工学部, 教授 (40012637) 太田隆久東京大学, 農学部, 教授 (30011844) 三浦謹一郎東京大学, 工学学, 教授 (30000227) 石井信一北海道大学, 薬学部, 教授 (90001031)
Keywords	蛋白質工学 / 遺伝子工学 / プロテア-ゼインヒビタ- / ズブチリシンインヒビタ- / 蛋白質のX線結晶解析 / 酵素-インヒビタ-相互作用
Research Abstract	本報告では、SSIは「放線菌のズブチリシンインヒビタ-」を、SMPIは「放線菌の金属プロテア-ゼインヒビタ-」を表す。放線菌の金属プロテア-ゼSGMPA及びSGMPBの高感度蛍光性基資を用いて、SSI人工変異体の両酵素に対する阻害物質定数を求めた(石井)。遺伝子工学的手法を用いてSSIの種々のアミノ酸残基を置換し、これら変異体の特性ならびにSSI遺伝子発現系の特性を明らかにした(三浦)。SSIに対するモノクロ-ナル抗体の作成を試み、数種のIgG1及びIgM産生クロ-ンを確立し、NMRによるエピト-プ解析を行った(荒田)。シンクロトロン放射光を用いてSSI及び変異型SSIとズブチリシンとの複合体の結晶のX線回折デ-タの収集に成功し、これに基づく構造精密化を行った(三井)。耐熱性プロテア-ゼ、アクアライシンIと野生型及び変異型SSIとの結合を解析し、本酵素の活性部位構造につき検討した(太田)。クロ-ン化した遺伝子を用いるSMPIの生産において、菌体外分泌生産量を増大する条件を検討した(高橋)。遺伝子工学的手法による部位特異的アミノ酸置換がSSIの高次構造に及ぼす効果を、安定同位体利用NMRにより解析した(甲斐荘)。SSI変異体につき重水素NMRを行い分子構造の「ゆらぎ」を解析した(赤坂)。SH/SS化合物の電気化学的徴量分析法を確立した(千田)。SSI変異体とズブチリシン及びSMPIとサ-モリシンの相互作用を平衡論的および速度論的に解析した(廣海・外村)。海洋細菌から新規ペプチド性セリンプロテア-ゼイヒビタ-、マリノスタチン、を単離し、一次構造を決定した(原)。ペプスタチン非感受性の酸性プロテア-ゼの新規インヒビタ-、チロスタチン,の構造を決定した(小田)。好熱菌から単離した新規耐熱性酸性プロテア-ゼが特徴ある性質を有し極めて高い基質特異性をもつことを明らかにした(村尾)。

Research Products

(25 results)

All Other

All Publications (25 results)

[Publications] Ichiro Terada: "Unique Precursor Structure of an Extracellular Protease,Aqualysin I.with N- and C-terminal Pro-sequences and Its Processing in Escherichia Coli" J.Biol.Chem.(1990)
[Publications] susumu Shibata: "Computer simulation of Proton Spin Relaxation in Proteins: Application to Streptomyces Subtilisin Inhibitor" Magn.Reson.in Chem.28. (1990)
[Publications] Koichi Kato: "Application of ^<13>C Nuclear Magnetic Resonance Spectroscopy to Molecular Structural Analysis of Antibody Molecules" J.Biochem.105. 867-869 (1989)
[Publications] Kohei Oda: "A Novel Proteinase Inhibitor,Tyrostatin,Inhibiting Some Pepstatin-insensitive Carboxyl Proteinases" Agric.Biol.Chem.53(2). 405-415 (1989)
[Publications] Y.Takeuchi: "Molecular recognition at the interface between subtilisin BPN´ and its Proteinaceous inhibitor SSI" Proceedings of the Second International Conference on Protein Engineering. (1989)
[Publications] Wataru Ito: "Protein-protein interactions on the surface of immunoglobulin molecules" J.Mol.Graphics. 7. 60-63 (1989)
[Publications] Seiichi Taguchi: "Analysis of transcriptional control regions in the Streptomyces subtilisin-inhibitor-encoding gene" Gene. 84. 279-286 (1989)
[Publications] Seiichi Taguchi: "Efficient extracellular expression of a foreign protein in Streptomyces using secretory protease inhibitor(SSI)gene fusions" Biotechnology. 7. 1063-1066 (1989)
[Publications] Shusei Obata: "Molecular Cloning and Nucleotide Seguence Determination of Gene Encoding Streptomyces Subtilisin Inhibitor(SSI)" J.Biochem.105. 367-371 (1989)
[Publications] Shusei Obata: "High-Level Expression in Streptomyces lividans 66 of a Gene Encoding Streptomyces Subtilisin Inhibitor from Streptomyces albogriseolus s-3253" J.Biochem.105. 372-376 (1989)
[Publications] K.Tanaka: "The coding and regulatory sequences of the metalloproteinase inhibitor(S-MPI)gene of Streptomyes nigrescens"
[Publications] H.Aoki: "Cloning and nucleotide sequencing of an extracellular protease gene from Streptomyces lividans"
[Publications] M.Kainosho: "Determination of Side-Chain Conformation of Amino Acid Residues in Protein.A Tailored Isotope Labeling Approach" J.Amer.Chem.Soc.
[Publications] T.Higashijima: "^<19>F-and ^<31>P-NMR Measurement of α-Subunit of GTP-binding Regulatory Proteins(G_0 and G_S)Activated by F^-"
[Publications] 村尾澤夫: "発茸誘導促進物質" 日農化誌. 63(4). 874-876 (1989)
[Publications] 村尾澤夫: "放線菌由来のメタルプロテイナ-ゼインヒビタ-" 蛋白質・核酸・酵素. 34(8). 980-988 (1989)
[Publications] 小島修一: "放線菌サブチリシンインヒビタ-の蛋白質工学" 蛋白質・核酸・酵素. 34(8). 939-948 (1989)
[Publications] 三浦謹一郎: "プロテア-ゼ・インヒビタ-設計のこころみ" BIOmedica. 5(3). 284-288 (1990)
[Publications] 竹内康雄: "タンパク質設計への道" 現代化学増刊. (1990)
[Publications] T.Kumazaki: "Primary Structure of Ascidian Trypsin Inhibitors in the Hemolymph of a Solitary Ascidian,Holocynthia roretzi" J.Biochem.107. (1990)
[Publications] T.Kumazaki: "Disulfide Bridge Structure of Ascidian Trypsin Inhibitor I:Similarity to Kazal-Type Inhibitors" J.Biochem.107. (1990)
[Publications] 梶原賢太: "ビマン系蛍光基質を用いたメタロプロテア-ゼの活性測定とこれによるSSIとの相互作用の解析" 生化学. 61. 834 (1989)
[Publications] 吉正佳代: "Streptomyces subtilisin inhibitor(SSI)の反応部位結合(Met73-Va174)のsubtilisin BPN´による修復速度" 生化学. 61. 983 (1989)
[Publications] 藤谷靖志: "放線菌メタルプロテア-ゼインヒビタ-(S-MPI)とサ-モリシンの相互作用" 日農化誌. 63(3). 620 (1989)
[Publications] Y.Takeuchi: "GBF Monographs"Advances in Protein Design"(Vol.12)" VCH Publishers, 177-183 (1989)

1989 Fiscal Year Annual Research Report

蛋白質工学的手法を活用した徴生物起源の蛋白性プロテア-ゼインヒビタ-の多角的研究

Principal Investigator

廣海 啓太郎 京都大学, 農学部, 名誉教授 (50025425)

Research Products

[Publications] Ichiro Terada: "Unique Precursor Structure of an Extracellular Protease,Aqualysin I.with N- and C-terminal Pro-sequences and Its Processing in Escherichia Coli" J.Biol.Chem.(1990)

[Publications] susumu Shibata: "Computer simulation of Proton Spin Relaxation in Proteins: Application to Streptomyces Subtilisin Inhibitor" Magn.Reson.in Chem.28. (1990)

[Publications] Koichi Kato: "Application of ^<13>C Nuclear Magnetic Resonance Spectroscopy to Molecular Structural Analysis of Antibody Molecules" J.Biochem.105. 867-869 (1989)

[Publications] Kohei Oda: "A Novel Proteinase Inhibitor,Tyrostatin,Inhibiting Some Pepstatin-insensitive Carboxyl Proteinases" Agric.Biol.Chem.53(2). 405-415 (1989)

[Publications] Y.Takeuchi: "Molecular recognition at the interface between subtilisin BPN´ and its Proteinaceous inhibitor SSI" Proceedings of the Second International Conference on Protein Engineering. (1989)

[Publications] Wataru Ito: "Protein-protein interactions on the surface of immunoglobulin molecules" J.Mol.Graphics. 7. 60-63 (1989)

[Publications] Seiichi Taguchi: "Analysis of transcriptional control regions in the Streptomyces subtilisin-inhibitor-encoding gene" Gene. 84. 279-286 (1989)

[Publications] Seiichi Taguchi: "Efficient extracellular expression of a foreign protein in Streptomyces using secretory protease inhibitor(SSI)gene fusions" Biotechnology. 7. 1063-1066 (1989)

[Publications] Shusei Obata: "Molecular Cloning and Nucleotide Seguence Determination of Gene Encoding Streptomyces Subtilisin Inhibitor(SSI)" J.Biochem.105. 367-371 (1989)

[Publications] Shusei Obata: "High-Level Expression in Streptomyces lividans 66 of a Gene Encoding Streptomyces Subtilisin Inhibitor from Streptomyces albogriseolus s-3253" J.Biochem.105. 372-376 (1989)

[Publications] K.Tanaka: "The coding and regulatory sequences of the metalloproteinase inhibitor(S-MPI)gene of Streptomyes nigrescens"

[Publications] H.Aoki: "Cloning and nucleotide sequencing of an extracellular protease gene from Streptomyces lividans"

[Publications] M.Kainosho: "Determination of Side-Chain Conformation of Amino Acid Residues in Protein.A Tailored Isotope Labeling Approach" J.Amer.Chem.Soc.

[Publications] T.Higashijima: "^<19>F-and ^<31>P-NMR Measurement of α-Subunit of GTP-binding Regulatory Proteins(G_0 and G_S)Activated by F^-"

[Publications] 村尾澤夫: "発茸誘導促進物質" 日農化誌. 63(4). 874-876 (1989)

[Publications] 村尾澤夫: "放線菌由来のメタルプロテイナ-ゼインヒビタ-" 蛋白質・核酸・酵素. 34(8). 980-988 (1989)

[Publications] 小島修一: "放線菌サブチリシンインヒビタ-の蛋白質工学" 蛋白質・核酸・酵素. 34(8). 939-948 (1989)

[Publications] 三浦謹一郎: "プロテア-ゼ・インヒビタ-設計のこころみ" BIOmedica. 5(3). 284-288 (1990)

[Publications] 竹内康雄: "タンパク質設計への道" 現代化学増刊. (1990)

[Publications] T.Kumazaki: "Primary Structure of Ascidian Trypsin Inhibitors in the Hemolymph of a Solitary Ascidian,Holocynthia roretzi" J.Biochem.107. (1990)

[Publications] T.Kumazaki: "Disulfide Bridge Structure of Ascidian Trypsin Inhibitor I:Similarity to Kazal-Type Inhibitors" J.Biochem.107. (1990)

[Publications] 梶原賢太: "ビマン系蛍光基質を用いたメタロプロテア-ゼの活性測定とこれによるSSIとの相互作用の解析" 生化学. 61. 834 (1989)

[Publications] 吉正佳代: "Streptomyces subtilisin inhibitor(SSI)の反応部位結合(Met73-Va174)のsubtilisin BPN´による修復速度" 生化学. 61. 983 (1989)

[Publications] 藤谷靖志: "放線菌メタルプロテア-ゼインヒビタ-(S-MPI)とサ-モリシンの相互作用" 日農化誌. 63(3). 620 (1989)

[Publications] Y.Takeuchi: "GBF Monographs"Advances in Protein Design"(Vol.12)" VCH Publishers, 177-183 (1989)

廣海啓太郎京都大学, 農学部, 名誉教授 (50025425)