Dataset Information

Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system.

ABSTRACT:

Background

Recently, 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.

Results

AtPPT1 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.

Conclusion

Formation 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.

SUBMITTER: Nozawa A

PROVIDER: S-EPMC3090341 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Publications

Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system.

Nozawa Akira A Ogasawara Tomio T Matsunaga Satoko S Iwasaki Takahiro T Sawasaki Tatsuya T Endo Yaeta Y

BMC biotechnology 20110411

<h4>Background</h4>Recently, 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.<h4>Results</h4>AtPPT1 was synthesized using a wheat cell-free syst ...[more]

PMID: 21481249

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Dataset Information

Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system.

Background

Results

Conclusion

Publications

Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system.

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