A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins
| Autor: | Satomi Suzuki, Toshihiko Utsumi, Keishi Aizawa, Nobuyuki Uozumi, Takahiro Hohsaka, Keietsu Abe, Masaaki Ito, Gunnar von Heijine, Satoshi Souma, Eiji Ando, Yoko Sato, Toru Ezure, Kei Nanatani |
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| Rok vydání: | 2014 |
| Předmět: |
Signal peptide
Insecta Structural Engineering lcsh:Medicine Bioengineering Biology Endoplasmic Reticulum Research and Analysis Methods Cell Line Twin-arginine translocation pathway Microsomes Animals Amino Acids Molecular Biology Techniques lcsh:Science Lipid bilayer Molecular Biology Integral membrane protein Multidisciplinary Cell-Free System Staining and Labeling lcsh:R Peripheral membrane protein Membrane Proteins Biology and Life Sciences Intracellular Membranes Translocon Transport protein Membrane protein Biochemistry Engineering and Technology lcsh:Q Research Article Biotechnology |
| Zdroj: | PLoS ONE, Vol 9, Iss 12, p e112874 (2014) PLoS ONE |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0112874 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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