1990 Fiscal Year Annual Research Report

ス-パ-シグナルペプチドの開発と有用タンパク質の分泌生産への応用

Research Project

Project/Area Number	02558018
Research Institution	The University of Tokyo
Principal Investigator	水島昭二東京大学, 応用微生物研究所, 教授 (50013313)
Co-Investigator(Kenkyū-buntansha)	疋田千波東京大学, 応用微生物研究所, 特別研究員 (60222238) 松山伸一東京大学, 応用微生物研究所, 助手 (50183108)
Keywords	タンパク質の分泌 / シグナルペプチド / ス-パ-シグナルペプチド / シグナルペプチド正荷電域 / シグナルペプチド疎水域 / 人工シグナルペプチド / 大腸菌 / タンパク質の膜透過
Research Abstract	1.N末端正荷電の最適化:シグナルペプチドのN末端正荷電数をゼロから4まで変化させると、正荷電数の増加に従って膜透過活性は増加する。しかしそれ以上の増加は活性増加につながらないようであった。正荷電数を8としたものを各種作成してみたが、それらでは透過活性はほとんど見られなかった。 2.中央疎水領域の最適化:疎水領域のポリペプチド鎖の長さとともに全疎水度の影響を調べるために、従来の〔Leu〕nシリ-ズに対して、〔Leu,Ala〕nシリ-ズの分泌型タンパク質を多数作成して、透過活性を測定した。〔Leu,Ala〕nシリ-ズでもアミノ酸残基数に対して高い特異性が示された。しかし最適の残基数は〔Leu〕nの場合より長かった。以上のことは、疎水領域は膜透過装置によって特異的に認識されていること、その認識には全疎水度も関与していることを示唆している。 3.C末端領域の最適化:シグナル切断部位近くにPro残基があるか否かによって膜過透のプロトン駆動力依存性がシグナルペプチド切断とは無関係に大きく変化することを明らかにした。シグナル切断領域の高次構造が膜透過に重要なかかわりあいをもっていることを示している。 4.各領域の組合せを含めた最適化:N末端領域の正荷電の膜透過活性への影響が疎水領域の長さによって変化することが分った。目下、〔Leu〕n,〔Leu,Ala〕nシリ-ズの変異体を用いてこの2つの領域間の機能的関係について解析を進めている。

Research Products

(11 results)

All Other

All Publications (11 results)

[Publications] Sasaki,S.,Matsuyama,S.and Mizushima,S.: "<In>___ー <vitro>___ー Kinetic analysis of the role of the positive charge at the aminoーterminal region of signal peptides in translocation of secretory protein across the cytoplasmic membrane in <Escherichia>___ー <coli.>___ー" J.Biol.Chem.265. 4358-4363 (1990)
[Publications] Matsuyama,S.,Kimura,E.and Mizushima,S.: "Complementation of two overlapping fragments of SecA,a protein translocation ATPase of <Escherichia>___ー <coli>___ー,allows ATP binding to its aminoーterminal region." J.Biol.Chem.265. 8760-8765 (1990)
[Publications] Akita,M.,Sasaki,S.,Matsuyama,S.and Mizushima,S.: "SecA interacts with secretory proteins by recognizing the positive charge at the aminoーterminus of the signal peptide in <Escherichia>___ー <coli>___ー." J.Biol.Chem.265. 8164-8169 (1990)
[Publications] Tokuda,H.,Kim,Y.J.and Mizushima,S.: "<In>___ー <vitro>___ー protein translocation into inverted membrane vesicles prepared from <Vibrio>___ー <alginolyticus>___ー is stimulated by the electrochemical potential of Na^+ in the presence of <Escherichia>___ー <coli>___ー SecA." FEBS Letters. 264. 10-12 (1990)
[Publications] Mizushima,S.and Tokuda,H.: "<In>___ー <vitro>___ー translocation of bacterial secretory proteins and energy requirements." J.Bioenerg.Biomemb.22. 389-399 (1990)
[Publications] Matsuyama,S.,Akimaru,J.and Mizushima,S.: "SecEーdependent overproduction of SecY in <Escherichia>___ー <coli>___ー:Evidence for interaction between two components of the secretory machinery." FEBS Letters. 269. 96-100 (1990)
[Publications] Shiozuka,K.,Tani,K.,Mizushima,S.and Tokuda,H.: "The proton motive force lowers the level of ATP required for the <in>___ー <vitro>___ー translocation of a secretory protein in <Escherichia>___ー <coli>___ー." J.Biol.Chem.265. 18843-18847 (1990)
[Publications] Tani,K.,Tokuda,H.and Mizushima,S.: "Translocation of proOmpA possessing an intramolecular disulfide bridge into membrane vesicles of <Escherichia>___ー <coli>___ー:Effect of membrane energization." J.Biol.Chem.265. 17341-17347 (1990)
[Publications] Tokuda,H.,Shiozuka,K.and Mizushima S.: "Reconstitution of translocation activity for secretory proteins from solubilized components of <Escherichia>___ー <coli>___ー." Eur.J.Biochem.192. 583-589 (1990)
[Publications] Shinkai,A.,Akita,M.,Matsuyama,S.and Mizushima,S.: "Quantitative renaturation from a guanidineーdenatured state of the SecA dimer,a 200 kDa protein involved in protein secretion in <Escherichia>___ー <coli>___ー." Biochem.Biophys.Res.Commun.172. 1217-1223 (1990)
[Publications] Yamane,K.,Akiyama,Y.,Ito,K.and Mizushima,S.: "A positively charged region is a determinant of the orientation of cytoplasmic membrane proteins in <Escherichia>___ー <coli>___ー." J.Biol.Chem.265. 21166-21171 (1990)

1990 Fiscal Year Annual Research Report

ス-パ-シグナルペプチドの開発と有用タンパク質の分泌生産への応用

Principal Investigator

水島昭二東京大学, 応用微生物研究所, 教授 (50013313)

Research Products

[Publications] Matsuyama,S.,Kimura,E.and Mizushima,S.: "Complementation of two overlapping fragments of SecA,a protein translocation ATPase of <Escherichia>_ー <coli>_ー,allows ATP binding to its aminoーterminal region." J.Biol.Chem.265. 8760-8765 (1990)

[Publications] Akita,M.,Sasaki,S.,Matsuyama,S.and Mizushima,S.: "SecA interacts with secretory proteins by recognizing the positive charge at the aminoーterminus of the signal peptide in <Escherichia>_ー <coli>_ー." J.Biol.Chem.265. 8164-8169 (1990)

[Publications] Mizushima,S.and Tokuda,H.: "<In>_ー <vitro>_ー translocation of bacterial secretory proteins and energy requirements." J.Bioenerg.Biomemb.22. 389-399 (1990)

[Publications] Matsuyama,S.,Akimaru,J.and Mizushima,S.: "SecEーdependent overproduction of SecY in <Escherichia>_ー <coli>_ー:Evidence for interaction between two components of the secretory machinery." FEBS Letters. 269. 96-100 (1990)

[Publications] Shiozuka,K.,Tani,K.,Mizushima,S.and Tokuda,H.: "The proton motive force lowers the level of ATP required for the <in>_ー <vitro>_ー translocation of a secretory protein in <Escherichia>_ー <coli>_ー." J.Biol.Chem.265. 18843-18847 (1990)

[Publications] Tani,K.,Tokuda,H.and Mizushima,S.: "Translocation of proOmpA possessing an intramolecular disulfide bridge into membrane vesicles of <Escherichia>_ー <coli>_ー:Effect of membrane energization." J.Biol.Chem.265. 17341-17347 (1990)

[Publications] Tokuda,H.,Shiozuka,K.and Mizushima S.: "Reconstitution of translocation activity for secretory proteins from solubilized components of <Escherichia>_ー <coli>_ー." Eur.J.Biochem.192. 583-589 (1990)

[Publications] Shinkai,A.,Akita,M.,Matsuyama,S.and Mizushima,S.: "Quantitative renaturation from a guanidineーdenatured state of the SecA dimer,a 200 kDa protein involved in protein secretion in <Escherichia>_ー <coli>_ー." Biochem.Biophys.Res.Commun.172. 1217-1223 (1990)

[Publications] Yamane,K.,Akiyama,Y.,Ito,K.and Mizushima,S.: "A positively charged region is a determinant of the orientation of cytoplasmic membrane proteins in <Escherichia>_ー <coli>_ー." J.Biol.Chem.265. 21166-21171 (1990)

1990 Fiscal Year Annual Research Report

ス-パ-シグナルペプチドの開発と有用タンパク質の分泌生産への応用

Principal Investigator

水島 昭二 東京大学, 応用微生物研究所, 教授 (50013313)

Research Products

[Publications] Matsuyama,S.,Kimura,E.and Mizushima,S.: "Complementation of two overlapping fragments of SecA,a protein translocation ATPase of <Escherichia>___ー <coli>___ー,allows ATP binding to its aminoーterminal region." J.Biol.Chem.265. 8760-8765 (1990)

[Publications] Akita,M.,Sasaki,S.,Matsuyama,S.and Mizushima,S.: "SecA interacts with secretory proteins by recognizing the positive charge at the aminoーterminus of the signal peptide in <Escherichia>___ー <coli>___ー." J.Biol.Chem.265. 8164-8169 (1990)

[Publications] Mizushima,S.and Tokuda,H.: "<In>___ー <vitro>___ー translocation of bacterial secretory proteins and energy requirements." J.Bioenerg.Biomemb.22. 389-399 (1990)

[Publications] Matsuyama,S.,Akimaru,J.and Mizushima,S.: "SecEーdependent overproduction of SecY in <Escherichia>___ー <coli>___ー:Evidence for interaction between two components of the secretory machinery." FEBS Letters. 269. 96-100 (1990)

[Publications] Shiozuka,K.,Tani,K.,Mizushima,S.and Tokuda,H.: "The proton motive force lowers the level of ATP required for the <in>___ー <vitro>___ー translocation of a secretory protein in <Escherichia>___ー <coli>___ー." J.Biol.Chem.265. 18843-18847 (1990)

[Publications] Tani,K.,Tokuda,H.and Mizushima,S.: "Translocation of proOmpA possessing an intramolecular disulfide bridge into membrane vesicles of <Escherichia>___ー <coli>___ー:Effect of membrane energization." J.Biol.Chem.265. 17341-17347 (1990)

[Publications] Tokuda,H.,Shiozuka,K.and Mizushima S.: "Reconstitution of translocation activity for secretory proteins from solubilized components of <Escherichia>___ー <coli>___ー." Eur.J.Biochem.192. 583-589 (1990)

[Publications] Shinkai,A.,Akita,M.,Matsuyama,S.and Mizushima,S.: "Quantitative renaturation from a guanidineーdenatured state of the SecA dimer,a 200 kDa protein involved in protein secretion in <Escherichia>___ー <coli>___ー." Biochem.Biophys.Res.Commun.172. 1217-1223 (1990)

[Publications] Yamane,K.,Akiyama,Y.,Ito,K.and Mizushima,S.: "A positively charged region is a determinant of the orientation of cytoplasmic membrane proteins in <Escherichia>___ー <coli>___ー." J.Biol.Chem.265. 21166-21171 (1990)

水島昭二東京大学, 応用微生物研究所, 教授 (50013313)

[Publications] Matsuyama,S.,Kimura,E.and Mizushima,S.: "Complementation of two overlapping fragments of SecA,a protein translocation ATPase of <Escherichia>_ー <coli>_ー,allows ATP binding to its aminoーterminal region." J.Biol.Chem.265. 8760-8765 (1990)

[Publications] Akita,M.,Sasaki,S.,Matsuyama,S.and Mizushima,S.: "SecA interacts with secretory proteins by recognizing the positive charge at the aminoーterminus of the signal peptide in <Escherichia>_ー <coli>_ー." J.Biol.Chem.265. 8164-8169 (1990)

[Publications] Mizushima,S.and Tokuda,H.: "<In>_ー <vitro>_ー translocation of bacterial secretory proteins and energy requirements." J.Bioenerg.Biomemb.22. 389-399 (1990)

[Publications] Matsuyama,S.,Akimaru,J.and Mizushima,S.: "SecEーdependent overproduction of SecY in <Escherichia>_ー <coli>_ー:Evidence for interaction between two components of the secretory machinery." FEBS Letters. 269. 96-100 (1990)

[Publications] Shiozuka,K.,Tani,K.,Mizushima,S.and Tokuda,H.: "The proton motive force lowers the level of ATP required for the <in>_ー <vitro>_ー translocation of a secretory protein in <Escherichia>_ー <coli>_ー." J.Biol.Chem.265. 18843-18847 (1990)

[Publications] Tani,K.,Tokuda,H.and Mizushima,S.: "Translocation of proOmpA possessing an intramolecular disulfide bridge into membrane vesicles of <Escherichia>_ー <coli>_ー:Effect of membrane energization." J.Biol.Chem.265. 17341-17347 (1990)

[Publications] Tokuda,H.,Shiozuka,K.and Mizushima S.: "Reconstitution of translocation activity for secretory proteins from solubilized components of <Escherichia>_ー <coli>_ー." Eur.J.Biochem.192. 583-589 (1990)

[Publications] Shinkai,A.,Akita,M.,Matsuyama,S.and Mizushima,S.: "Quantitative renaturation from a guanidineーdenatured state of the SecA dimer,a 200 kDa protein involved in protein secretion in <Escherichia>_ー <coli>_ー." Biochem.Biophys.Res.Commun.172. 1217-1223 (1990)

[Publications] Yamane,K.,Akiyama,Y.,Ito,K.and Mizushima,S.: "A positively charged region is a determinant of the orientation of cytoplasmic membrane proteins in <Escherichia>_ー <coli>_ー." J.Biol.Chem.265. 21166-21171 (1990)