Change search
ReferencesLink to record
Permanent link

Direct link
A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins
Show others and affiliations
2014 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 9, no 12, e112874- p.Article in journal (Refereed) Published
Abstract [en]

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.
National Category
Biological Sciences
URN: urn:nbn:se:su:diva-113574DOI: 10.1371/journal.pone.0112874ISI: 000346907600012OAI: diva2:786388


Available from: 2015-02-05 Created: 2015-02-04 Last updated: 2015-02-05Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
von Heijine, Gunnar
By organisation
Department of Biochemistry and Biophysics
In the same journal
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 23 hits
ReferencesLink to record
Permanent link

Direct link