title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Cell-Free Science and Technology Research Center and the Venture Business, Laboratory, Ehime University, 3 Bunkyo-Cho, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Nozawa
authors.forename: Tomio
authors.initials: T
authors.lastname: Ogasawara
authors.forename: Satoko
authors.initials: S
authors.lastname: Matsunaga
authors.forename: Takahiro
authors.initials: T
authors.lastname: Iwasaki
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sawasaki
authors.forename: Yaeta
authors.initials: Y
authors.lastname: Endo
mesh_terms: D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014908:Triticum / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: D029681:Arabidopsis Proteins; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C481826:phosphate translocator 1, Arabidopsis; D049452:Green Fluorescent Proteins