Cell free expression in proteinosomes prepared from native protein-PNIPAAm conjugates

Towards the goal of building synthetic cells from the bottom-up, the establishment of micrometer-sized compartments that contain and support cell free transcription and translation that couple cellular structure to function is of critical importance. Proteinosomes, formed from crosslinked cationized protein-polymer conjugates offer a promising solution to membrane-bound compartmentalisation with an open, semi-permeable membrane. Critically, to date, there have been no demonstration of cell free transcription and translation within water-in-water proteinosomes. Herein, we present a novel approach to the fabrication of proteinosomes directly from native protein-polymer (BSA-PNIPAAm) conjugates. We show that these native proteinosomes offer an excellent alternative as artificial cell chassis. Significantly, the native proteinosomes are stable under high salt conditions and can consequently support cell free transcription and translation. The native proteinosomes offer enhanced protein expression compared to proteinosomes prepared from traditional methodologies. Furthermore, we demonstrate the integration of proteinosomes into higher order cellular architectures with membrane free compartments and liposomes. The integration of bioinspired architectural elements with the central dogma is an essential building block for realizing minimal synthetic cells and is key for exploiting artificial cells in real-world applications.

从底层构建合成细胞（synthetic cells）的目标出发，建立可容纳并支撑无细胞转录与翻译（cell-free transcription and translation）、将细胞结构与功能相耦合的微米级区室，具有至关重要的意义。由交联阳离子化蛋白-聚合物偶联物（crosslinked cationized protein-polymer conjugates）形成的蛋白体（Proteinosomes），为具备开放半透性膜的膜结合区室化方案提供了极具前景的解决路径。迄今为止，尚无关于水包水型蛋白体（water-in-water proteinosomes）内实现无细胞转录与翻译的研究报道。本文提出一种直接以天然蛋白-聚合物偶联物（BSA-PNIPAAm）制备蛋白体的创新方法。研究表明，这类天然蛋白体可作为性能优异的人工细胞底盘（artificial cell chassis）。尤为关键的是，天然蛋白体在高盐环境下稳定性优异，因而可支撑无细胞转录与翻译过程；相较于传统方法制备的蛋白体，该天然蛋白体可实现更高水平的蛋白质表达。此外，本研究还证明可将蛋白体与无膜区室、脂质体（liposomes）整合为更高阶的细胞架构。将仿生结构元件与中心法则（central dogma）相结合，是实现极简合成细胞的核心构建基础，也是推动人工细胞走向实际应用的关键所在。