A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins
2014 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 9, no 12, e112874- p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2014. Vol. 9, no 12, e112874- p.
IdentifiersURN: urn:nbn:se:su:diva-113574DOI: 10.1371/journal.pone.0112874ISI: 000346907600012OAI: oai:DiVA.org:su-113574DiVA: diva2:786388