Abstract
BackgroundRecently, some groups have reported on cell-free synthesis of functional membrane proteins (MPs) in the presence of exogenous liposomes (liposomes). Previously, we reported synthesis of a functional AtPPT1 plant phosphate transporter that was associated with liposomes during translation. However, it is unclear whether or not lipid/MP complex formation is common to all types of MPs in the wheat cell-free system.ResultsAtPPT1 was synthesized using a wheat cell-free system with or without liposomes. AtPPT1 synthesized with liposomes showed high transport activity, but the activity of AtPPT1 synthesized without liposomes was less than 10% activity of that with liposomes. To test whether co-translational association with liposomes is observed in the synthesis of other MPs, we used 40 mammalian MPs having one to 14 transmembrane domains (TMDs) and five soluble proteins as a control. The association rate of all 40 MPs into liposomes was more than 40% (mean value: 59%), while that of the five soluble proteins was less than 20% (mean value: 12%). There were no significant differences in association rate among MPs regardless of the number of TMDs and synthesis yield. These results indicate that the wheat cell-free system is a highly productive method for lipid/MP complex formation and is suitable for large-scale preparation. The liposome association of green fluorescent protein (GFP)-fusion MPs were also tested and recovered as lipid/MP complex after floatation by Accudenz density gradient ultracentrifugation (DGU). Employment of GFP-MPs revealed optimal condition for Accudenz floatation. Using the optimized Accudenz DGU condition, P2RX4/lipid complexes were partially purified and detected as a major band by Coomassie Brilliant Blue (CBB)-staining after SDS-PAGE.ConclusionFormation of lipid/AtPPT1 complex during the cell-free synthesis reaction is critical for synthesis of a functional MP. The lipid/MP complex during the translation was observed in all 40 MPs tested. At least 29 MPs, as judged by their higher productivity compared to GFP, might be suitable for a large-scale preparation. MPs synthesized by this method form lipid/MP complexes, which could be readily partially purified by Accudenz DGU. Wheat cell-free protein synthesis in the presence of liposomes will be a useful method for preparation of variety type of MPs.
Highlights
Some groups have reported on cell-free synthesis of functional membrane proteins (MPs) in the presence of exogenous liposomes
Timing of liposome-supplementation to wheat cell-free translation system for synthesis of functional MPs Previously, we reported synthesis and liposome association of functional MPs using a wheat cell-free system supplemented with liposomes [16]
To verify that cotranslational association of MP with liposomes is critical for functional synthesis, we tested the synthesis of an Arabidopsis thaliana phosphate translocator, AtPPT1, in the presence of, absence of, and after post-translational addition of liposomes
Summary
Some groups have reported on cell-free synthesis of functional membrane proteins (MPs) in the presence of exogenous liposomes (liposomes). We reported synthesis of a functional AtPPT1 plant phosphate transporter that was associated with liposomes during translation. It is unclear whether or not lipid/MP complex formation is common to all types of MPs in the wheat cell-free system. Goren and Fox [15] showed reconstitution of the functional stearoyl Co-A desaturase complex, which consists of three proteins, cytochrome b5, cytochrome b5 reductase, and human stearoyl-CoA desaturase 1 (hSCD1) synthesized by wheat cell-free system in the presence of asolectin liposomes. The general versatility of this method is unclear as the above examples focus on specific MPs
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