Neuroscience Program, University of Michigan, Ann Arbor; Cellular and Molecular Biology Program, University of Michigan, Ann Arbor.
Department of Mechanical Engineering, University of Michigan, Ann Arbor.
J Vis Exp. 2024 Mar 8(205). doi: 10.3791/66595.
Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.
无细胞表达 (CFE) 系统是合成生物学中的强大工具，可在合成细胞中模拟生物感应和能量再生等细胞功能。然而，要重建广泛的细胞过程，就需要将膜蛋白成功地重建到合成细胞的膜中。虽然在常见的 CFE 系统中可溶性蛋白的表达通常是成功的，但将膜蛋白在合成细胞的脂质双层中进行重建被证明是具有挑战性的。在这里，展示了一种基于封装和在合成细胞内孵育 CFE 反应，将模型膜蛋白细菌谷氨酸受体 (GluR0) 再构成巨大的单室囊泡 (GUVs) 作为模型合成细胞的方法。利用该平台，展示了用蛋白酶紫质信号肽替代 GluR0 的 N 端信号肽对 GluR0 成功共翻译转位到杂交 GUV 膜中的影响。该方法提供了一种稳健的程序，可将各种膜蛋白在合成细胞中进行无细胞再构成。
