1991 Fiscal Year Final Research Report Summary
Creation of Novel Membrane Proteins by Manipulation of Sorting Signal Sequences.
| Project/Area Number |
01880020
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
物質生物化学
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| Research Institution | Kyushu University |
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Principal Investigator |
OMURA Tsuneo Kyushu Unibersity, Graduate School of Medical Science, Professor, 大学院医学系研究科, 教授 (80029933)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIBASHI Tsuyoshi Fukuoka Industrial Technology Center, Investigator, 専門研究員
MIHARA Katsuyoshi Kyushu University, Graduate School of Medical Science, Associate Professor, 大学院医学系研究科, 助教授 (40029963)
ITO Akio Kyushu University, Faculty of Science, Professor, 理学部, 教授 (30037379)
KUNITAKE Toyoki Kyushu University, Faculty of Engineering, Professor, 工学部, 教授 (40037734)
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| Project Period (FY) |
1989 – 1991
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| Keywords | Membrane protein / Sorting Signal Sequence / Biomembrane / Artificial bilayer membrane |
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| Research Abstract |
1. Analysis of membrane-targeting sorting signal sequences.
Characterization and Analysis of the sorting sequences which target proteins cotranslationally to endoplasmic reticulum (ER) membrane were carried out. Structural requirements for the amino-terminal signal sequences and signal-anchor seuqences were examined with a cell-free system, and it was concluded that the balance between the charges at the amino-terminus and the length of the hydrophobic stretch determines the function of the sequences. Structural requirements for the function of stop-transfer sequences were also examined with a cell-free cotranslational system, and the decisive importance of the length of the hydrophobic stretch in the arrest of peptide translocation across ER membrane was confirmed. The carboxy-terminal sorting sequences, that are functional in the post-translational membrane integration of several ER membrane proteins and Mt outer membrane proteins were also characterized and analyzed.
2. Expression of
… More
modified membrane proteins In animal cells and microbial cells.
A new lipofection method of introducing DNAs Into cultured mammalian cells was established by the use of synthetic amphipathic organic compounds. Modified membrane proteins having altered sorting sequences were successfully expressed with several types of cultured cells including COS1 cells, and their Intracellular localization was studied. Expression of the modified membrane proteins an yeast cells and Escherichia coli cells was also studied in detail.
3. Synthesis of amphipathic organic compounds for artificial bilayer membranes.
Various types of amphipathic organic compounds resembling phospholipids were synthesized, and the formation of bilayer membranes In aqueous solutions was examined. The physicochemical properties of the bimolecular layer membranes were studied.
4. Incorporation of proteins into the artificial bilayer membranes.
Incorporation of water soluble proteins, Including myoglobin and peroxidase, Into the hydrophilic layer of the artificial bilayer membrane of synthetic organic compounds was achieved. The incorporated proteins were stable even after the membrane film was taken out from the aqueous medium and dried. Judging from the orientation of the protoheme in the membrane, the incorporated proteins have a fixed uniform orientation in the membrane. Less
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