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.
Highlights
Membrane proteins constitute almost one third of all gene products in any type of organism
In order to deliver functional membrane proteins to the endoplasmic reticulum (ER) membrane, it is necessary that a cell-free translation/translocation system preserve the integrity of the involved pathways
For the isolation of extracts containing the translational machinery from cultured Spodoptera frugiperda 21 (Sf21) cells a modified method based on a method for isolation of rough microsomes from dog pancreas was used [13, 14]
Summary
Membrane proteins constitute almost one third of all gene products in any type of organism. Because membrane proteins are embedded in the cell membrane they are in direct contact with the outside of the cell and are major targets for pharmaceutical or physiological regulation. For this reason there is frequently a need to be able to produce these proteins in the laboratory. A cell-free translation/translocation system is the preferred method to expedite the production of a membrane protein of interest. A novel protein-targeting pathway, the guided entry of tail anchored proteins (GET) pathway, that directs the targeting machinery for tail-anchored membrane proteins (TA-proteins) to the ER membrane, has been described [2]. In order to deliver functional membrane proteins to the ER membrane, it is necessary that a cell-free translation/translocation system preserve the integrity of the involved pathways
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