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A Cell-Free Translo...
A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins
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Ezure, Toru (författare)
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Nanatani, Kei (författare)
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Sato, Yoko (författare)
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Suzuki, Satomi (författare)
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Aizawa, Keishi (författare)
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Souma, Satoshi (författare)
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Ito, Masaaki (författare)
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Hohsaka, Takahiro (författare)
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- von Heijine, Gunnar (författare)
- Stockholms universitet,Institutionen för biokemi och biofysik
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Utsumi, Toshihiko (författare)
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Abe, Keietsu (författare)
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Ando, Eiji (författare)
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Uozumi, Nobuyuki (författare)
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(creator_code:org_t)
- 2014-12-08
- 2014
- Engelska.
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:12, s. e112874-
- Relaterad länk:
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https://doi.org/10.1...
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https://journals.plo...
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.
Ämnesord
- NATURVETENSKAP -- Biologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences (hsv//eng)
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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PLOS ONE
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- Av författaren/redakt...
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Ezure, Toru
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Nanatani, Kei
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Sato, Yoko
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Suzuki, Satomi
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Aizawa, Keishi
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Souma, Satoshi
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visa fler...
-
Ito, Masaaki
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Hohsaka, Takahir ...
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von Heijine, Gun ...
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Utsumi, Toshihik ...
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Abe, Keietsu
-
Ando, Eiji
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Uozumi, Nobuyuki
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visa färre...
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- NATURVETENSKAP
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NATURVETENSKAP
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och Biologi
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PLOS ONE
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Stockholms universitet