A method of gentle hydration to prepare oil-free giant unilamellar vesicles that can confine enzymatic reactions

K. Shohda, K. Takahashi, A. Suyama

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6 Scopus citations

Abstract

We report a new and improved method to prepare, by gentle hydration of lipid films, oil-free giant unilamellar vesicles (GUVs), in which enzymatic reactions can be encapsulated. The traditional method of gentle hydration requires very low concentrations of metal ions, whereas enzymatic reactions generally require mono- and divalent metal ions at physiological concentrations. In order to improve the production of oil-free GUVs that can confine enzymatic reactions, we developed a novel method also based on gentle hydration, but in which the precursor lipid film was doped with both 1,2-dioleoyl-. sn-glycero-3-phosphoethanolamine-. N-[methoxy(polyethylene glycol)-2000] (PEGylated lipid) and sugar. Close examination of the size, shape, and lamellarity of vesicles prepared in this manner demonstrated that the process improves the production of oil-free GUVs even at low temperatures and physiological salt concentrations. PEGylated lipid and sugar were found to synergistically improve GUV formation. Finally, we demonstrate the successful enzymatic synthesis of RNA within oil-free GUVs that were prepared on ice.

Original languageEnglish (US)
Pages (from-to)76-82
Number of pages7
JournalBiochemistry and Biophysics Reports
Volume3
DOIs
StatePublished - Sep 1 2015

Bibliographical note

Funding Information:
This work was supported by Grants-in-Aid for Scientific Research on Innovative Areas ( 24104001-5 to K.S., 23119007 to A.S.) from The Ministry of Education, Culture, Sports, Science, and Technology , Japan.

Publisher Copyright:
© 2015 The Authors.

Keywords

  • Flow cytometry
  • GUV
  • Gentle hydration
  • Liposome
  • Membrane protein
  • Synthetic biology

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