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

• Published in: PloS one 2014-12, Vol.9 (12), p.e112874-e112874
• Main Authors: Ezure, Toru, Nanatani, Kei, Sato, Yoko, Suzuki, Satomi, Aizawa, Keishi, Souma, Satoshi, Ito, Masaaki, Hohsaka, Takahiro, von Heijine, Gunnar, Utsumi, Toshihiko, Abe, Keietsu, Ando, Eiji, Uozumi, Nobuyuki
• Format: Article
• Language: English
• Subjects: Amino acids; Amino Acids - chemistry; Amino Acids - metabolism; Animal tissues; Animals; Biology and Life Sciences; Cell Line; Cell-Free System; Endoplasmic Reticulum - metabolism; Engineering and Technology; Glycosylation; Insect cells; Insecta; Insects; Intracellular Membranes - metabolism; Machinery and equipment; Membrane lipids; Membrane proteins; Membrane Proteins - genetics; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Membranes; Microsomes; Microsomes - metabolism; Post-translation; Proteases; Protein biosynthesis; Protein synthesis; Protein transport; Proteins; Research and Analysis Methods; Spodoptera frugiperda; Staining and Labeling; Structure-function relationships; Tails; Translocation
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0112874

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.