A cell-free translocation system using extracts of cultured insect cells to yield functional membrane proteins

Toru Ezure, Kei Nanatani, Yoko Sato, Satomi Suzuki, Keishi Aizawa, Satoshi Souma, Masaaki Ito, Takahiro Hohsaka, Gunnar Von Heijine, Toshihiko Utsumi, Keietsu Abe, Eiji Ando, Nobuyuki Uozumi

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

Abstract

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.

Original languageEnglish
Article numbere112874
JournalPloS one
Volume9
Issue number12
DOIs
Publication statusPublished - 2014 Dec 8

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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