TY - JOUR
AU1 - Ezure, Toru
AU2 - Nanatani, Kei
AU3 - Sato, Yoko
AU4 - Suzuki, Satomi
AU5 - Aizawa, Keishi
AU6 - Souma, Satoshi
AU7 - Ito, Masaaki
AU8 - Hohsaka, Takahiro
AU9 - von Heijine, Gunnar
AU1 - Utsumi, Toshihiko
AU1 - Abe, Keietsu
AU1 - Ando, Eiji
AU1 - Uozumi, Nobuyuki
AB - 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.
TI - A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins
JF - PLoS ONE
DO - 10.1371/journal.pone.0112874
DA - 2014-12-08
UR - https://www.deepdyve.com/lp/pubmed-central/a-cell-free-translocation-system-using-extracts-of-cultured-insect-aSSkzo56f0
SP - e112874
VL - 9
IS - 12
DP - DeepDyve
ER -