title: Cell-free protein synthesis inside giant unilamellar vesicles analyzed by flow cytometry.
abstract: Lipid vesicles have been used as model cell systems, in which an in-vitro transcription-translation system (IVTT) is encapsulated to carry out intravesicular protein synthesis. Despite a large number of previous studies, a quantitative understanding of how protein synthesis inside the vesicles is affected by the lipid membrane remains elusive. This is mainly because of the heterogeneity in structural properties of the lipid vesicles used in the experiments. We investigated the effects of the phospholipid membrane on green fluorescent protein (GFP) synthesis occurring inside cell-sized giant unilamellar vesicles (GUV), which have a defined quantity of lipids relative to the reaction volume. We first developed a method to distinguish GUV from multilamellar vesicles using flow cytometry (FCM). Using this method, we investigated the time course of GFP synthesis using one of the IVTT, the PURE system, and found that phospholipid in the form of GUV has little effect on GFP synthesis based on three lines of investigation. (1) GFP synthesis inside the GUV was not dependent on the size of GUV (2) or on the fraction of cholesterol or anionic phospholipid constituting the GUV, and (3) GFP synthesis proceeded similarly in GUV and in the test tube. The present results suggest that GUV provides an ideal reaction environment that does not affect the internal biochemical reaction. On the other hand, we also found that internal GFP synthesis is strongly dependent on the chemical composition of the outer solution.
authors.affiliation: Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Nishimura
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Matsuura
authors.forename: Kazuya
authors.initials: K
authors.lastname: Nishimura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sunami
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Suzuki
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yomo
mesh_terms: D002474:Cell-Free System / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002784:Cholesterol / Q000737:chemistry; D004339:Drug Compounding; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000737:chemistry; D007700:Kinetics; D010316:Particle Size; D010713:Phosphatidylcholines / Q000737:chemistry*; D014176:Protein Biosynthesis / Q000187:drug effects; D053835:Unilamellar Liposomes / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D010713:Phosphatidylcholines; D053835:Unilamellar Liposomes; D049452:Green Fluorescent Proteins; D002784:Cholesterol