Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
Institute for Auditory Neuroscience, University Medical Center Göttingen, Göttingen, Germany.
Sci Rep. 2018 Jun 21;8(1):9422. doi: 10.1038/s41598-018-27456-4.
Reconstitution of membrane proteins in artificial membranes is an essential prerequisite for functional studies that depend on the context of an intact membrane. While straight-forward protocols for reconstituting proteins in small unilamellar vesicles were developed many years ago, it is much more difficult to prepare large membranes containing membrane proteins at biologically relevant concentrations. Giant unilamellar vesicles (GUVs) represent a model system that is characterised by low curvature, controllable tension, and large surface that can be easily visualised with microscopy, but protein insertion is notoriously difficult. Here we describe a convenient method for efficient generation of GUVs containing functionally active SNARE proteins that govern exocytosis of synaptic vesicles. Preparation of proteo-GUVs requires a simple, in-house-built device, standard and inexpensive electronic equipment, and employs a straight-forward protocol that largely avoids damage of the proteins. The procedure allows upscaling and multiplexing, thus providing a platform for establishing and optimizing preparation of GUVs containing membrane proteins for a diverse array of applications.
在人工膜中重建膜蛋白是功能研究的必要前提,这些功能研究依赖于完整膜的环境。虽然多年前就已经开发出了用于在小单层囊泡中重组蛋白的简单方案,但要制备含有生物相关浓度的膜蛋白的大膜则要困难得多。巨大的单层囊泡(GUV)是一种模型系统,其特点是曲率低、张力可控且表面积大,很容易用显微镜观察到,但蛋白质插入却非常困难。在这里,我们描述了一种方便的方法,可以有效地生成含有控制突触小泡胞吐作用的功能性 SNARE 蛋白的 GUV。制备蛋白-GUV 需要一个简单的、内部构建的设备、标准且廉价的电子设备,并采用一种简单的方案,在很大程度上避免了蛋白质的损伤。该方法允许扩大规模和多重化,从而为建立和优化含有膜蛋白的 GUV 的制备提供了一个平台,可用于各种应用。