Shohda K, Takahashi K, Suyama A
Department of Life Sciences and Institute of Physics, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
Biochem Biophys Rep. 2015 Jul 9;3:76-82. doi: 10.1016/j.bbrep.2015.07.005. eCollection 2015 Sep.
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--glycero-3-phosphoethanolamine--[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.
我们报告了一种新的改进方法，通过脂质膜的温和水合作用来制备无油巨型单层囊泡（GUVs），其中可以封装酶促反应。传统的温和水合方法需要极低浓度的金属离子，而酶促反应通常需要生理浓度的单价和二价金属离子。为了提高能够限制酶促反应的无油GUVs的产量，我们开发了一种同样基于温和水合作用的新方法，但在前体脂质膜中同时掺杂了1,2 - 二油酰 - sn -甘油 - 3 - 磷酸乙醇胺 - [甲氧基（聚乙二醇）- 2000]（聚乙二醇化脂质）和糖。对以这种方式制备的囊泡的大小、形状和片层性进行仔细检查表明，该方法即使在低温和生理盐浓度下也能提高无油GUVs的产量。发现聚乙二醇化脂质和糖能协同促进GUV的形成。最后，我们展示了在冰上制备的无油GUVs内成功进行的RNA酶促合成。
